Assemblies and methods for reducing optical crosstalk in particle processing systems

ABSTRACT

The present disclosure relates to optical crosstalk reduction in particle processing (e.g., cytometry including flow cytometry using microfluidic based sorters, drop formation based sorters, and/or cell purification) systems and methods in order to improve performance. More particularly, the present disclosure relates to assemblies, systems and methods for minimizing optical crosstalk during the analyzing, sorting, and/or processing (e.g., purifying, measuring, isolating, detecting, monitoring and/or enriching) of particles (e.g., cells, microscopic particles, etc.). The exemplary systems and methods for crosstalk reduction in particle processing systems (e.g., cell purification systems) may be particularly useful in the area of cellular medicine or the like. The systems and methods may be modular and used singly or in combination to optimize cell purification based on the crosstalk environment and specific requirements of the operator and/or system.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 61/784,431, titled “Assemblies and Methods forReducing Optical Crosstalk in Particle Processing Systems,” and filedMar. 14, 2013, the content of which is hereby incorporated by referencein its entirety.

TECHNICAL FIELD

The present disclosure relates to crosstalk reduction, and particularly,to reducing optical crosstalk in particle processing systems.

BACKGROUND

In general, particle processing (e.g., cytometry) systems (e.g.,cytometers) and methods are known. For example, some approaches toparticle processing or analyzing (e.g., cell purification) systems suchas sorting flow cytometers and other particle processing systems haveproven to be useful in life science research, industrial, agricultural,diagnostics, and other medical applications.

In general, a cytometer can be described as a system that can measurelarge numbers of homogeneous and/or heterogeneous particle sets toachieve statistically relevant data sets that can be used to groupand/or identify subpopulations that reside within a given particlepopulation (e.g., within one or more samples). These measurements aresometimes performed optically (whether they are intrinsic or responsiveto an optical stimulus), or they may be electrical in nature (or someother physical, chemical, or biological characteristic) as a stream ofparticles passes through a measurement or inspection zone. The particlesets may include biological entities such as cells (e.g., bacteria,viruses, organelles, yeasts, spores, genetic material, spermatozoa, eggcells, multicellular organisms), or other organisms, or other naturallyoccurring or synthetic/synthetically derived objects.

With the addition of sort functionality, a cytometer can also be used toisolate (e.g., physically separate) one or more particles of interestfrom a given sample through operator control. See, e.g., U.S. Pat. No.6,248,590, the entire content of which is hereby incorporated byreference in its entirety. In general, this technique can be used toclassify and/or separate (e.g., purify or enrich) one or morepopulations as defined by the operator.

Cell purification means, such as flow cytometry, can be used to processmicroscopic particles of biological interest, such as cells or viruses,based on optical properties of the particles. However, when multiplesensors are in use, there exists the possibility that attributiveinterference or optical crosstalk may occur, which can limit the abilityto provide broad accurate dynamic measurement ranges for the sensinglocations and/or particles of interest.

For example, it is desired that any light emanating from one particlesensing location should not interfere with the light being measured fromanother particle sensing location. If there is any such opticalcrosstalk interference, then some measurements made may be erroneous,and the further data analysis and/or further processing steps (such asproducing a diagnostic assessment, or separation based on measuredand/or differentiated characteristics) are likely to be affected.

Some measured characteristics of particles positioned at one sensinglocation may then mask or be masked by characteristics of anotherparticle at another location. An example of this effect may be seen whena very low-response (e.g. dimly fluorescent) particle is to be measuredwhile a particle with bright fluorescence happens to be within anadditional sensing location within a similar timeframe. As anon-limiting example, if there is potential for optical crosstalk withina given system, the dim particle may be erroneously measured as beingbrighter than it actually is, since an amount of light signal emanatingfrom the bright particle may also be captured.

The likelihood of such optical crosstalk may be increased when there isclose proximity of sensing locations, or when particles are located onthe same substrate or sensing region, or other common opticalcomponentry or light paths. Thus, measurement accuracy may becompromised, which could cause issues in diagnostic applications, e.g.,where critical treatment decisions are based on such measurements.

Additionally, in cell purification applications and cell sorting, sucherroneous measurements may restrict the ability to provide suitablesub-populations with suitable purity, recovery, and/or yield, sinceunwanted particles or cells may be inadvertently separated based oninaccurate particle classification. It is therefore desirable to havesystems and methods for reducing such optical crosstalk in particleanalysis systems and/or cell purification systems.

SUMMARY

The present disclosure provides crosstalk reduction in particleprocessing (e.g., microfluidic based sorters, drop formation basedsorters, and/or cell purification) systems and methods in order toimprove performance. More particularly, the present disclosure relatesto assemblies, systems and methods for minimizing optical crosstalkduring the analyzing, sorting, and/or processing of particles (e.g.,cells, microscopic particles, etc.).

The present disclosure provides signal processing systems (e.g., lightexcitation or illumination systems, on-chip aspects, light collectionand detection systems, combined optical and electronic systems(excitation and collection/detection systems)) designed to minimizecrosstalk and methods of using such systems. In certain embodiments, thepresent disclosure relates to the processing and/or measurement ofparticles within a microfluidic system where multiple sensors areemployed. In other embodiments, the present disclosure relates to theprocessing of particles utilizing drop based sorters or the like.

As such, one embodiment of the present disclosure is directed generallyto a signal processing system including a multi-element photodetectorassembly or array (e.g., a CCD array, CMOS array, photodiode arrayphotomultiplier tube (PMT) array) which senses signals from multipleparticle sensing locations and one or a plurality of wavebands. Incertain embodiments, the signal processing system includes a lightcollection system which senses and processes optical signals. In certainembodiments, the light collection system includes an array, the arrayfurther including a dichroic block, a detector, and a scrambled fiberbundle disposed therebetween (e.g., disposed between the dichroic blockand the detector). The system may further include image plane confocalapertures, a single lens system and a microfluidic chip array withillumination apertures. The system may further include a plurality offlow channels or paths provided by capillaries, cuvettes, and/or nozzlesthat may form one or more fluid streams, jets and/or droplets.

The present disclosure is further directed to a method of using a signalprocessing system including a light collection system which minimizesoptical crosstalk. The method may comprise the following steps combinedor in the alternative: separating the flow channels; using large spanoptical systems for excitation and collection; using spatial filterswhich employ pinholes on-chip and off chip near object, image, or nearFourier planes; using isolated optical pick-up systems, where light froma plurality of particle (sensing) locations is collected by an opticalsystem; and/or using scrambled light mapping, which may be spatial orspectral in nature.

Another embodiment of the present disclosure is a signal processingsystem characterized by its ability to perform spatial and/or temporalmodulation at the level of illumination source, or the detector, or theuse of heterodyne detection by providing an electronic oscillatorgenerator and a local oscillator array in the event of stationaryinterference; or when confocal properties are desired, by providing anelectronic oscillator generator and a pulse generator.

Another embodiment of the present disclosure is a method for signalmodulation.

The present disclosure also provides for a modular system that can bepackaged as a kit of components for particular applications in cellularmedicine or the like.

The present disclosure provides for a particle processing systemincluding a particle processing region; a signal processing system incommunication with the particle processing region; wherein the signalprocessing system is configured and adapted to reduce crosstalk betweena plurality of signal paths to improve performance of the particleprocessing system. The signal processing system may be an optical signalprocessing system adapted to reduce optical crosstalk between aplurality of optical paths.

The present disclosure also provides for a signal processing system tominimize crosstalk of a particle processing system including: a signalprocessing system that maps signals emanating from a plurality ofparticle sensing locations to a plurality of detector or sensorlocations; wherein the mapping alters the order of the signal paths sothat signals from adjacent particle sensing locations are reorganized sothat they are no longer adjacent at the sensing locations.

The present disclosure also provides for a signal processing system tominimize optical crosstalk of a particle processing system including: anoptical system that maps optical signals emanating from a plurality ofparticle sensing locations to a plurality of photodetector orphotosensor locations; wherein the mapping alters the order of opticalpaths so that light from adjacent particle sensing locations is alteredso that they are no longer adjacent at photosensing locations. Thepresent disclosure also provides for a light processing system tominimize optical crosstalk of a particle processing system, wherein theoptical system mapping uses optical fibers or a fiber bundle. Thepresent disclosure also provides for a light processing system tominimize optical crosstalk of a particle processing system, wherein theoptical system mapping uses minors or steering elements.

The present disclosure also provides for a method of using a particleprocessing system comprising the following steps combined or in thealternative: separating flow channels; using large span optical systemsfor excitation and collection; using spatial filters which employpinholes on-chip and off chip near object, image, or Fourier planes;using isolated optical pick-up systems, where light from a plurality ofparticle locations is collected by the optical system; and usingscrambled light mapping, which may be spatial or spectral in nature.

The present disclosure also provides for a optical processing system toreduce optical crosstalk in a particle processing system, the opticalprocessing system including: a multi-element photo-multiplier tubearray; wherein the multi-element photo-multiplier tube array isconfigured and adapted to reduce optical crosstalk to improveperformance of the particle processing system. The present disclosurealso provides for a optical processing system to reduce opticalcrosstalk in a particle processing system further including: a dichroicblock, a detector, and a scrambled fiber bundle disposed between thedichroic block and the detector; image plane confocal apertures; asingle lens system; and a microfluidic chip array with illuminationapertures. The present disclosure also provides for an opticalprocessing system to reduce optical crosstalk in a particle processingsystem, wherein the scrambled fiber bundle is a strategically mappedfiber bundle to photodetector sensor scheme that minimizes opticalcrosstalk.

In exemplary embodiments, the present disclosure provides for a systemhaving at least some of the following elements/features:

1) illumination or excitation (e.g., where this can be parallel orsimultaneous, scanned, switched or pulsed, involve apertures, etc.,);

2) plurality of particle illumination or sensing locations (spacing orproximity to each other, blocking features between each, metal layers orapertures, etc.);

3) optical collection of light (including apertures, imaging systems,lenses, reflective elements, diffractive elements, etc.);

4) spectral selection elements (e.g., including optical filters such asdichroic, neutral density, longpass, bandpass, shortpass or combinationsthereof);

5) optical delivery or steering methods (such as scrambling that couldbe carried out using fibers or other optical elements); and/or

6) photodetection (single or plurality depending on scheme), which mayalso include electronic techniques such as those used to time excitationor detection of particles relative to a particular sensing location at aparticular time.

Any combination or permutation of embodiments is envisioned. Additionaladvantageous features, functions and applications of the disclosedsystems, assemblies and methods of the present disclosure will beapparent from the description which follows particularly when read inconjunction with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the present disclosure are further describedwith reference to the appended figures. It is to be noted that thevarious features and combination of features described below andillustrated in the figures can be arranged and/organized differently toresult in embodiments which are still within the spirit and scope of thepresent disclosure. To assist those of ordinary skill in the art inmaking and using the disclosed systems, assemblies and methods,reference is made to the appended figures, wherein:

FIGS. 1A-1O depict exemplary features of particle processing systemsaccording to the present disclosure;

FIG. 2 depicts an exemplary particle processing system according to thepresent disclosure;

FIGS. 3A-3E depict other exemplary particle processing systems, methodsand data according to the present disclosure;

FIG. 4 depicts another exemplary particle processing systems, methodsand data according to the present disclosure;

FIG. 5 depicts another exemplary particle processing systems, method anddata according to the present disclosure;

FIGS. 6A-6B depict other exemplary particle processing systems, methodsand data according to the present disclosure;

FIGS. 7A-7B depict other exemplary particle processing systems, methodsand data according to the present disclosure;

FIGS. 8A-8D depict other exemplary particle processing systems, methodsand data according to the present disclosure;

FIGS. 9A-9D depict other exemplary particle processing systems, methodsand data according to the present disclosure;

FIG. 10 depicts another exemplary particle processing system, methodsand data according to the present disclosure;

FIGS. 11A-11B depict other exemplary particle processing systems,methods and data according to the present disclosure;

FIGS. 12A-12B depict other exemplary particle processing systems,methods and data according to the present disclosure; and

FIG. 13 depicts another exemplary particle processing systems, methodsand data according to the present disclosure.

DETAILED DESCRIPTION

In the description which follows, like parts are marked throughout thespecification and drawings with the same reference numerals,respectively. Drawing figures are not necessarily to scale and incertain views, parts may have been exaggerated for purposes of clarity.

DEFINITIONS

Crosstalk: can mean, but is in no way limited to, unwanted signalartifacts that enter an signal path and lead to errors in particle orcell measurement, characterization, and/or purification.

Scrambled: can mean, but is in no way limited to, interlaced orinterweaved, and/or random or purposefully arranged, and/or out ofsequential order.

The present disclosure provides improved optical crosstalk reduction inparticle processing (e.g., microfluidic based sorters, drop formationbased sorters, and/or cell purification) systems and methods. Ingeneral, the present disclosure provides for assemblies, systems andmethods for minimizing optical crosstalk during the analyzing, sorting,processing, and/or purifying of particles (e.g., cells, microscopicparticles, etc.), thereby providing a significant commercial and/oroperational advantage as a result. In certain embodiments, the presentdisclosure provides a light mapping apparatus and method for highsensitivity (optical) measurement of a plurality of microfluidicchannels. In certain embodiments, the present disclosure provides alight mapping apparatus and method for high sensitivity (optical)measurement of a plurality particle flow paths produced by capillaries,nozzles, or jets.

The exemplary systems and methods for crosstalk reduction in particleprocessing systems (e.g., cell purification systems) may be particularlyuseful in the area of cellular medicine or the like, and/or cell orparticle sorting applications (e.g., industrial cell or particle sortingapplications, such as, for example, yeast, veterinary (e.g., sperm),life sciences research and/or applications, etc.). The systems andmethods may be modular and used singly or in combination to optimizecell purification based on the crosstalk environment and specificrequirements of the operator and system.

In exemplary embodiments, the present disclosure provides assemblies,systems and methods to ensure accurate measurement of optical signalsproduced by particles from multiple locations on a microfluidic cellpurification system by reducing optical crosstalk. The term “crosstalk”is to be understood to encompass unwanted signal artifacts that enter asignal path and lead to errors in particle or cell measurement,characterization, and/or purification. These particles may be containedwithin or through one or more flow channels, where it is necessary tomeasure such objects at two or more spatially separated locations,and/or where measurement errors due to crosstalk must be minimized. Itis noted that accuracy in purification is of paramount importance,particularly in the area of cellular medicine (e.g., applicationsrequiring optical sensitivity) and related applications, and/or cell orparticle sorting applications (e.g., industrial cell or particle sortingapplications, such as, for example, yeast, veterinary (e.g., sperm),life sciences research and/or applications, etc.).

The source of such errors can be related to: (i) optical sources thatinteract with the microfluidic chip and/or particles of interest; (ii)the optical properties of the surrounding material (such as chip scatteror auto-fluorescence as non-limiting examples); (iii) the presence orabsence of other particles or debris in close proximity to the particleof interest (e.g., whether in the same or different flow channels,whether stationary or moving); (iv) properties of optical systems thatare used to collect and/or transport light emanating from or interactingwith light sourced from light sources used (e.g., lenses, minors,optical fibers, filters); and/or (v) other considerations such as thesensing method (e.g., sensor type, geometry, spatial separation, opticalproperties). Additionally, the system may also be applied to non-flowapplications but where there is the potential for erroneous measurementsdue to crosstalk and the like (e.g., optical crosstalk arising fromoptical imaging systems). For example, the exemplary systems may enablehigh resolution imaging of cells, other particles and/or microscopicobjects to be performed.

If one or more particles are to measured simultaneously or nearsimultaneously, there is a chance that unwanted light that is destinedfor one sensor as related to one particle, ends up contaminating (e.g.,adding to) the light that is destined for another sensor. Even in theabsence of a second particle while a first particle is sensed, there maybe unwanted light from other locations that ends up being directed to afirst sensor that is related (e.g., matched) to the first particle. Thisunwanted signal may be (scattered) excitation (e.g. laser, LED,monochromatic, polychromatic, etc.), fluorescence, or other light.

In exemplary embodiments, systems and methods are described herein thatreduce the potential for erroneous and/or artificial measurement ofparticles by employing techniques that minimize unwanted signals frominteracting with wanted signals. In general, the systems may utilize anumber of approaches in order to reduce unwanted crosstalk. The presentdisclosure successfully addresses the problem of measuring anddifferentiating particles, especially dimly fluorescent particles, assensed from more than one location, whether mobile or stationary. Inparticle analysis systems that have a large number of sensing locations,or multiple inspection points, it is desired to reduce the complexity,size, and cost of components, in general, and the optical and sensingcomponents, in particular.

When many sensors are required, there is a significant chance thatcrosstalk may occur, thus limiting the ability to provide a broaddynamic measurement range for all sensing locations and particle locatedat the various sensing locations.

However, discrete photodetectors that provide sufficient performance(e.g., speed, gain, sensitivity, noise, etc.) can be bulky, expensive,etc. To overcome these issues, a multi-element photodetection system(e.g., a multi-element photomultiplier tube) has been advantageouslydeveloped in order to sense optical signals from not one, but multiplesensor locations (e.g., micro-cytometers or sorters) and multiplewavebands of light for each microcytometer. For example, in oneembodiment, electronic mapping of the sensor pixels has been arranged ina certain manner for electronic design purposes.

In exemplary embodiments, it has been determined that electronic mappingof the sensor pixels be arranged in a certain manner for electronicdesign purposes. After further consideration of the design,calculations, and measurements, it was discovered that a surprisingadvantage of such an arrangement was that potential issues of crosstalkcan be mitigated to a great extent by employing a scattered or scrambledsensor location e.g., utilizing a light collection and transmission pathand carefully mapping these transmission paths to a plurality of sensorpixel positions. One illustrative example is using fibers that caneasily be moved relative to one another and using them for scrambledlight mapping (e.g., mapping these transmission paths to a plurality ofsensor pixel positions). This enables a reliable and robustartifact-free measurement and differentiation of particles.

In general, some systems provide for the measurement of particles withina microfluidic system where multiple sensors are employed. The presentdisclosure provides assemblies and methods to provide accuratemeasurement of optical signals produced by particles (or otherspecimens) from multiple locations on a microfluidic system or device.Particles may be defined as nano, micro, or macro-scopic objects (e.g.,including atoms, viruses, proteins, organisms, organic or inorganicobjects, cells, organelles, microarray spots, metals). In exemplaryembodiments, the present disclosure relates to the reliable and robustmeasurement of particles that flow through optical measurementassemblies and devices.

The particles may be contained within or through one or more flowchannels, where there is a desire to measure such objects at two or morespatially separated locations, and where there is a desire to minimizeerroneous or artificial measurements due to optical interference,scatter, fluorescence, phosphorescence, crosstalk, etc. (e.g., lightcapture from something other than the particle of interest that can ornot be differentiated from the light that is directly related to thatparticle).

In exemplary embodiments, the systems and methods of the presentdisclosure enable or facilitate the reliable and/or robust substantiallyartifact free and/or minimized measurement and differentiation ofparticles or other specimens or samples (whether flowing or not). Ingeneral, the particles are likely to be located at different points. Forthe case of a flow-through system, there may be two or more flow pathsor channels through which particles travel and are sensed (e.g., oftenas they interact with an excitation source).

As noted, some exemplary techniques for minimizing the potential forlight interference when measuring particles from more than one locationinclude: (i) excitation (e.g., scan and/or modulate excitation source,separate excitation source by spreading particle sensing locations);(ii) spatial filters (e.g., in front of excitation source to restrictlight from reaching unwanted areas, near objects and at or near object,Fourier, and image planes to reduce field of view of light collectionsystem thereby further minimizing unwanted light from reaching sensors);(iii) isolated optical collection (e.g., multiple optical collectionsystems and/or paths where the number of paths is less than the numberof prescribed particle locations, two particle locations might have twooptical collection systems or four particle locations may have two orfour optical collection systems); and/or (iv) scrambled (e.g.,interlaced/interweaved, random or purposefully arranged) light mapping(using optical fibers or other optical elements such as reflective,refractive, or diffractive elements).

In exemplary embodiments, by scanning and only having one sensing regionilluminated (and/or the detection on or enabled), then there should belittle or no interference with, or detectable signal from, other sensingregions.

For example, spatial filters (e.g., on the illumination side) limitunwanted light from reaching sensing location (and objects or structurearound sensing location) to minimize stray illumination or scattereffects. On the sensing side, spatial filters can provide the necessarykeyhole ability to only allow light from a particular region of interest(e.g., the sensing area of interest) to reach sensors or detectors,therefore minimizing stray light from non-target sensing areas fromreaching the detectors intended for measurement from target sensinglocations.

Moreover, isolated optical collection minimizes any chance of opticalcrosstalk on the light collection and detection side of the opticalsystem, also lessening the chance of internal scatter, autofluorescence,etc. from one optical channel (e.g., for one sensing location) frominterfering with another (e.g., optical channel and related sensinglocation).

Furthermore, scrambled fibers (i.e., out of sequential order) (whenlight can be mapped from a plurality of sensing locations to a pluralityof optical fibers), the light can be isolated, and purposefullytransmitted to pre-defined locations for detection that minimize anyfurther optical crosstalk related to detector geometry or size. Further,the particular spectral content of light, for the case of multiplefluorescent wavebands of interest, can be further interlaced to projectand isolate the light particular sensing locations and wavelengths frominterfering with light from other sensing locations or wavelengths.

In certain embodiments, the present disclosure provides techniques thatmaximize particle measurement accuracy in a multi-sensor particlemeasurement and sorting apparatus. In particular, it is desired that formulti-flow path systems that optical cross talk is minimized. Theexemplary systems/methods may also be applied to multiple sensinglocations for a single flow path (or static sample measurement system),or multiple sensing locations (e.g., more than one per) for multipleflow channel systems.

As such, exemplary embodiments of the present disclosure provide for theanalyzing, monitoring, and/or processing (e.g. sorting, ablating,modifying) of particles. Some potential advantageous uses of theexemplary systems/methods include those instances where some particlecharacteristics may be weak (e.g. small or low-interaction/transparentparticles), or uses that cover a broad dynamic range of response wheresensitivity of measurement and therefore insensitivity to opticalcrosstalk becomes important.

An example of this importance can be when it is desired that a verylow-response (e.g. dim fluorescence) particle is to be measured while aparticle with bright fluorescence happens to be within an additionalsensing location within a similar time-frame. If there is anysubstantial crosstalk in the system, the dim particle may be erroneouslymeasured as being brighter than it actually is. Thus, measurementaccuracy is compromised (which could cause issues in diagnosticapplications), and for sorting or other processing steps may result inthe particle being acted upon in some manner (e.g. sorted or not sorted)that does not accurately represent what should have occurred if themeasurement error due to crosstalk were not encountered.

Some alternative embodiments of the present disclosure include variousoptical isolation techniques that involve either temporally or spatiallyisolating illumination paths or geometries, sensing locations, lightcollection paths or geometries, and/or detector layouts to isolate light(and light wavelength) from a plurality of sensing locations (e.g.,scanning approaches, oscillating illumination or detection techniques,spectral un-mixing, separate sensor to sensor displacement).

It is also noted that one may use the scrambled or randomized approachinto tapered fiber bundles (rather than the non-scrambled approach) toproduce randomized light signals for security reasons as an example, orto take a light profile (Gaussian, laser as an example) and then producea uniform illumination area from the output of a 2D array of fibers(rather than a Gaussian output).

The present disclosure will be further described with respect to thefollowing non-limiting examples. These examples illustrate the systemsand methods of the present disclosure of improved optical crosstalkreduction in particle processing systems and methods.

In exemplary embodiments, the particle processing system may be amicrofluidic flow sorter particle processing system configured,dimensioned and adapted for analyzing, sorting, and/or processing (e.g.,purifying, measuring, isolating, detecting, monitoring and/or enriching)particles (e.g., cells, microscopic particles, etc.) or the like.However, it is noted that the systems and methods described may beapplied to other particle processing systems.

In certain embodiments, the particle processing system may be acytometer and/or a cell purification system or the like, although thepresent disclosure is not limited thereto. In exemplary embodiments, thesystem may be a microfluidic flow sorter particle processing system(e.g., microfluidic chip based system or drop formation particleprocessing system) or the like. Exemplary microfluidic flow sorterparticle processing systems and components or the like are disclosed,for example, in U.S. Pat. Nos. 8,277,764; 8,123,044; 7,569,788;7,492,522 and 6,808,075; and U.S. Patent Publication Nos. 2012/0009025;2012/0277902; 2011/0196637 and 2009/0116005; and U.S. Patent ApplicationSer. Nos. 61/647,821 and 61/702,114, the foregoing being incorporatedherein by reference in their entireties.

In further exemplary embodiments, the particle processing system may bea multi-channel or multi-jet flow sorter particle processing system(e.g., multiple capillaries or multiple fluid jet based systems) or thelike. Exemplary multi-channel or multi-jet flow sorter particleprocessing systems and components or the like are disclosed, forexample, in U.S. Patent Publication No. 2005/0112541, the entirecontents of which is hereby incorporated by reference in its entirety.

In exemplary embodiments, the present disclosure provides for a systemand method having at least some of the following elements, features,and/or steps for light mapping: (i) excitation (e.g., scanlaser/modulate laser (spatial and/or temporal); (ii) separate flowchannels (and use large span optical systems for excitation andcollection); (iii) spatial filters (pinholes on-ship and off chip nearobject, image or Fourier planes); (iv) isolated optical pick-up systems;(v) scrambled light mapping techniques (e.g., using fibers); and/or (vi)modulation (interlaced or high frequency switching and/or lock-indetection or sensing).

In certain embodiments, the present disclosure provides for a lightcollection system and method having at least some of the followingelements, features, and/or steps: (i) simultaneous excitation(excitation source on more than one particle), including separate flowcells and spatial filters (object plane and/or image plane); (ii)isolated optical collection (e.g., light from a plurality of particlelocations collected by optical system); and/or (iii) scrambled lightmapping (spatial and/or spectral).

In certain embodiments, the present disclosure provides for a modulationsystem and method having at least some of the following elements,features, and/or steps:

(i) scan source, including:

-   -   1) angular scan source across chip,    -   2) angular scan across a minor element (e.g., segmented mirror)        that reflects part or all of the source being scanned across        chip    -   3) scan slit/blocker across source,    -   4) pulse laser (delay),    -   5) pulse individual illumination channels (e.g., all on but        timing used to scan),    -   6) pulse laser (delay),    -   7) use speed of light to differentiate between channels (with        high speed detector), but take fluorescence lifetime into        account,    -   8) use lock-in detection,    -   9) spatial pattern/wavelet discrimination, and/or    -   10) scan detection pinholes;

(ii) scan detection, including:

-   -   1) scan pinholes,    -   2) optically addressable detection filter (scanning detection        blocker),    -   3) switch detector, including (a) power (single detector per        fluidic channel and/or single detector all fluidic        channels), (b) gain (single detector per fluidic channel and/or        single detector all fluidic channels), (c) electronics (single        detector per fluidic channel and/or single detector all fluidic        channels);

(iii) homodyne, including:

-   -   1) optical homodyne (coherent light),    -   2) balanced optical homodyne (coherent light),    -   3) electronic homodyne (incoherent light); and

(iv) heterodyne, including:

-   -   1) optical heterodyne (coherent light),    -   2) balanced optical heterodyne (coherent light),    -   3) electronic heterodyne (incoherent light).

In exemplary embodiments, scanning across a minor element (e.g.,segmented mirror that reflects part or all of the source being scannedacross chip) can minimize the dwell (e.g., dead or non-useful scan) timebetween channels and/or time not on the channels, therefore maximizingthe illumination/excitation time on channels/particle flow paths.

FIG. 1A illustrates the various subsystems and/or components that maygenerally be included in a particle processing system 100 according toembodiments presented herein. FIG. 1A schematically shows that particleprocessing system 100 may include a radiation source system 200, aradiation beam control system 300, a particle processing region 400, anemission signal collection system 500, a signal relay system 600, asignal conditioning system 700, a signal detection system 800 and anelectronics system 900.

Radiation source system 200 (or illumination source system) provides oneor more beams 205 of electromagnetic radiation. Radiation source system200 may include a single radiation source 210 or multiple radiationsources. Radiation source system 200 may also include beam shapingoptics 220 as are known in the art. One or more radiation beams 205exits the radiation source system 200 and enter the radiation beamcontrol system 300. A radiation source may also be referred to as anexcitation and/or illumination source and radiation beams may also bereferred to as excitation and/or illumination beams. Radiation refers toelectromagnetic radiation of any wavelength; similarly, light refers toelectromagnetic radiation of any wavelength. Radiation sources mayinclude lasers, LEDs, arc lamps, incandescent sources, radioactivesources, etc. Beam shaping optics may include refractive, reflective,diffractive, birefringent elements, etc. and any beam shaping, beamcombining and/or beam splitting elements.

Radiation beam control system 300 controls how one or more interrogationbeams 215 exiting the radiation beam control system impinge on orilluminate interrogation sites 155 provided by an interrogation element150. The radiation beam control system 300 may control the interrogationbeam(s) 215 spatially, temporally, and/or spectrally. Further, theradiation beam control system 300 may direct or manipulate the radiationbeam(s) dynamically and/or passively. As one non-limiting example,radiation beam control system 300 may include a spatial filter 310. Asother non-limiting examples, radiation beam control system 300 mayinclude an optical scanner 320, other scanners, electro-opticalmodulators, acousto-optical modulators, galvanic ormicro-electro-mechanical systems (MEMS)-based scanning optical elements,amplitude modulators, phase modulators, frequency modulators, etc.

The one or more interrogation beams 215 may be directed by radiationbeam control system 300 to illuminate a particle processing region 400.The particle processing region 400 may include a plurality ofinterrogation sites 155 a, 155 b, 155 c, etc. within an interrogationelement 150. The interrogation element 150 may be provided as part of amicrofluidic chip system 402.

Particle processing region 400 may be provided at the input side of thesignal collection system 500. As described in more detail below, theparticle processing region 400 may be a microfluidic chip system 402including a plurality of microfluidic flow channels 422. Particles ofinterest may travel through these microfluidic flow channels. Thesemicrofluidic flow channels may define the interrogation element 150 thatis exposed to one or more interrogation beams 215 for interrogating theparticles. Microfluidic chip system 402 may be provided with specificillumination apertures or masking patterns. Microfluidic chip system 402may include with a holder for ease of handling and for interfacing withother components of the particle processing system 100. Particleprocessing region 400 may alternatively be provided with one or morecapillaries, cuvettes, nozzles, cassettes, wells, reservoirs, etc.

Emission signals 505 are signals that are emitted from the interrogationelement 150. These emission signals 505 may be due to excitation,transmission, scatter, fluorescence, extinction, reflection, refraction,diffraction, etc. and are not limited as to any specific source (e.g.,particles, cells, edges, opaque regions, etc.). Emission signals 505,which include the signals of interest, are collected by emission signalcollection system 500 and transmitted to signal detection system 800 viaa signal relay system 600 and/or a signal conditioning system 700.

An emission signal collection system 500 may include a lens system 520to shape, focus and/or direct the emission signals 505. The lens system520 may be provided as a single lens system and/or as an array of lenssystems. According to some embodiments, the lens systems 520 may includea set of free optics to collect and/or reimage light or other signalsemitted from the interrogation element 150. For example, fluorescencesignals emitted from particles excited by an interrogation beam 215 maybe focused onto a fluorescence image plane. An example lens system(e.g., 520) may have a numerical aperture of 0.5. The same and/or otherlens systems may be used collect other emission signals 505 emanatingfrom the interrogation element 150, for example, extinction and/orscatter signals associated with particles. Further, emission signalcollection system 500 may include filters, whether spatial, spectral,long pass, short pass, band pass, etc. Emission signal collection system500 transmits collection signals 515 to signal relay system 600.

Signal relay system 600 may include a fiber bundle 620 (e.g., aplurality of optical fibers) for transmitting signals 605. This fiberbundle 620 may be provided between, and optically coupled to, emissionsignal collection system 500 and signal conditioning system 700.

Alternatively and/or additionally (as for example shown in FIG. 2) afiber bundle 620 may be provided between, and optically coupled to,signal conditioning system 700 and signal detection system 800. As willbe described in more detail below, according to certain embodiments, thefiber bundle 620 may be provided as a spatially scrambled fiber bundle,as a spectrally scrambled fiber bundle, or as a spatially and spectrallyscrambled fiber bundle.

Signal conditioning system 700 may include spatial filters (such asmasks 710 having apertures), spectral filters, dichroic arrays 720, etc.As non-limiting examples, signal conditioning system 700 may includelong pass filters, short pass filters, band pass filters, notch filters,absorptive elements, interference elements, polarization elements,spectral dispersion elements, etc.

Signal detection system 800 receives detector input signals 805 andconverts these into electrical signals 905 for transmission toelectronics 900. The signal detection system 800 may include a singledetector 810 or a plurality of detectors, for example photomultipliertubes (PMT), charge collection devices (CCD), avalanche photodiodes(APD), photodiodes, thermopiles, bolometers, etc. The detectors may bearranged as an array of detectors 820. Further, each detector 810 may beprovided as one or more sensors.

Electronics system 900 may be configured to acquire, process,characterize, and/or analyze the electrical signals emitted from thesignal detection system 800 and/or to control the particle processingsystem 100. The electrical signals 905 may be analog or digital.

The above-defined systems of the particle processing system 100 mayfurther define a signal processing system 1000. Signal processing system1000 may be in communication with one or more of the radiation sourcesystem 200, the radiation beam control system 300, the particleprocessing region 400, the emission signal collection system 500, thesignal relay system 600, the signal conditioning system 700, the signaldetection system 800, the electronics system 900, and/or portionsthereof. The signal processing system 1000 may create, acquire,manipulate, process, transmit, eliminate, augment, etc. signals(including electromagnetic, electrical, acoustic, optical, etc.) thatare involved in processing the particles in the particle processingsystem 100.

Further, in accord with some embodiments, certain of the above-describedsystems may be merged with another system, split between one or moreother systems, positioned elsewhere in the optical path, duplicatedand/or eliminated. Thus, for example, radiation beam control system 300may be eliminated or subsumed into radiation source system 200, in whichcase radiation beam(s) 205 and interrogation beam(s) 215 may be one andthe same. As another example, signal conditioning system 700 may beeliminated or subsumed into emission signal collection system 500 oralternatively into signal detection system 800, in which case signals605 exiting from signal relay system 600 and detector input signals 805may be one and the same.

FIG. 1B illustrates various modes that may be used to excite orinterrogate the particles P flowing through (or positioned within)interrogation sites 155 of the interrogation element 150. Theinterrogation element 150 may be located within a particle processingregion 400 which may include one or more microfluidic flow channels,wells, chambers, etc. (not shown). Thus, according to certainembodiments, the plurality of particles P1, P2 being illuminated and/orexcited may be flowing within a single microfluidic channel orpositioned within a single portion (such as a well or chamber) of theparticle processing region 400. Alternatively and/or additionally, theexcited particles P1, P2 may be flowing within two or more of themicrofluidic flow channels, wells, chambers, etc. As schematicallyillustrated in FIG. 1B(i), a plurality of particles P1, P2 may besimultaneously excited by a single interrogation beam 215 (or by aplurality of interrogation beams 215 a, 215 b). The single interrogationbeam 215 may be generated by a single radiation source 200 or may begenerated by a plurality of radiation sources 210 a, 210 b having theirradiation beams 205 a, 205 b combined into a single interrogation beam215. Optionally, a plurality of interrogation beams 215 a, 215 b may begenerated by a single radiation source 200 having its radiation beam 205split into the plurality of interrogation beams 215 a, 215 b or may begenerated by a plurality of radiation sources 210 a, 210 b.

As shown schematically in FIGS. 1B(ii) and 1B(iii), a particle P or aplurality of particles P1, P2 in the interrogation element 150 may benon-simultaneously excited (for example, sequentially) by one or moreinterrogation beams 215 a, 215 b. FIG. 1B(ii) shows that one or moreinterrogation beams 215 a, 215 b may sequentially or alternativelyinterrogate the particle(s) P. For example, first and secondinterrogation beams 215 a, 215 b may be alternatively pulsed so as tosequentially excite first and second particles P1, P2 (or tosequentially excite a single particle P). Optionally (not shown), theremay be only a single interrogation beam 215 and a means for selectivelyblocking the interrogation beam from interrogating both particles P1, P2at the same time may be provided. FIG. 1B(iii) shows that a plurality ofparticles P1, P2 may be sequentially or non-simultaneously excited orilluminated by an interrogation beam 215 that selectively scans or movesover the interrogation element 150. Relative to the excitation mode ofFIG. 1B(i), the excitation modes of FIGS. 1B(ii) and 1B(iii) generallylessen potential crosstalk problems.

FIG. 1C schematically illustrates that, at a very basic level, opticalcrosstalk may be eliminated or lessened by increasing the distance Dbetween the particles P being interrogated. Thus, increased distance D2between particles P1, P2 (and/or between flow paths within which theparticles travel) as shown in FIG. 1C(ii) as compared to a lesserdistance D1 (i.e., closer proximity) between particles P1, P2 (and/orbetween flow paths within which the particles travel) as shown in FIG.1C(i), may result in less crosstalk. For example, the amount of overlapbetween interrogation beams 215 a, 215 b, the amount of opticalinteraction between particles and/or adjacent material, and the amountof optical interaction of signals being collected for detection may allbe reduced by increasing the spacing or distance D between the particlesP. Unfortunately, competing design considerations generally precludespacing the particles far enough apart to completely eliminatecrosstalk.

FIG. 1D(i) shows a portion of a first microfluidic chip's substrate 420a provided with three microfluidic flow channels 422 arranged in arelatively low density channel spacing H1; FIG. 1D(ii) shows a portionof a second microfluidic chip's substrate 420 b provided with fivemicrofluidic flow channels 422 arranged over the same width, but with arelatively high density channel spacing H2.

FIG. 1E schematically illustrates that optical crosstalk may bemitigated by spatially filtering the interrogation beam(s) 215interrogating the particles P1, P2 (FIG. 1E(i)) and/or by spatiallyfiltering the emission signals 505 exiting the interrogation element 150(FIG. 1E(ii)). Spatial filters include angular filters. In FIG. 1E(i), aspatial filter or mask 310 is shown located between the interrogationbeam 215 and the interrogation element 150. Mask 310 is provided withone or more apertures 312 a, 312 b that only allow energy from theinterrogation beam(s) 215 to reach interrogation element 150 at specificlocations. In FIG. 1E(ii), a spatial filter or mask 710 is shown locatedbetween the interrogation element 150 and the signal detection system800. Mask 710 is provided with one or more apertures 712 a, 712 b thatonly allow energy emitted from the particles P1, P2 or otherinterrogated components to reach signal detection system 800 fromspecific locations of the interrogation element 150. For certainembodiments, these locations on or in the interrogation element 150 maycoincide with the flow channels 420 of the particle processing region400 and more specifically, may coincide with the particles P flowingthrough the channels. According to other embodiments, spatial filters ormasks may be positioned at or near the interrogation element 150, at ornear a Fourier plane of any optical collection system, and/or at or nearan image plane.

In an example embodiment, FIGS. 1F(i) and 1F(ii) show a portion of amicrofluidic chip's substrate 420 with microfluidic flow channels 422 a,422 b and a spatial filter 410 located at or near the detection plate150. Filter 410 is shown as mask formed as a metal layer provided on thesubstrate 420 and extending over the flow channels 422 a, 422 b. Thus,in this particular embodiment, spatial filter 410 is associated with theparticle processing region 400. If mask 410 is provided on theexcitation side of the interrogation element 150, then the apertures 412a, 412 b (shown as pairs of apertures in this particular embodiment)allow restricted portions of the interrogation beams to illuminatewithin the flow channels 422. If mask 410 is provided on the emissionside of the interrogation element 150, then the apertures 412 a, 412 ballow restricted portions of the emission signals to radiate from withinthe flow channels 422.

FIG. 1G schematically illustrates a spatial filter 510 associated withan embodiment of an emission signal collection system 500. Microfluidicchannels 422 a, 422 b are positioned at interrogation element 150 withina microfluidic chip's substrate 420. Emission beams 505 a, 505 b emanatefrom within microfluidic channels 422 a, 422 b, respectively, and arecollected by lens system 520 of the emission signal collection system500. Collection signals 515 a and 515 b, respectively associated withemission beams 505 b, 505 a and channels 422 b, 422 a, are spatiallyfiltered by mask 510. Mask 510 has a confocal aperture at 512 a thatallows collection signal 515 a to pass through to the signal detectionsystem 800 (e.g., an optical detection element). Mask 510 blockscollection signal 515 b at 514. Thus, in the embodiment of FIG. 1G,spatial filter 510 is associated with the emission signal collectionsystem 500 and further, the spatial filter 510 is used to mask detectedlight downstream of the lens system 520.

FIG. 1H schematically illustrates two alternative lens systems 520. FIG.1H(i) illustrates a single lens systems 520 a that collects emissionsignals 505 a, 505 b from a plurality of particle locations. Lens system520 a may have a plurality of optical elements (not shown) which shape,focus, direct, etc. emission signals 505 a, 505 b into collectionsignals 515 b, 515 a. Collection signals 515 a, 515 b may be focused atfocal plane 516. A spatial filter 510 allows emission from the particles(or specific locations associated with the interrogation element 150) toreach lens system 520 a through apertures 512 a, 512 b and blocks allother emissions. Similarly in FIG. 1H(ii), a spatial filter 510 allowsemission from the particles (or specific locations associated with theinterrogation element 150) to reach lens system 520 through apertures512 a, 512 b and blocks all other emissions. However, in the embodimentof FIG. 1H(ii) the lens system 520 includes a first lens system 520 cand a second lens system 520 d. Each lens system 520 c, 520 d may have aplurality of optical elements (not shown) which shape, focus, direct,etc. emission signals 505 c, 505 d into collection signals 515 c, 515 c.The collection signals 515 c, 515 d may be focused at focal plane 516. Aspatial filter (not shown) may be provided at focal plane 516. In FIG.1H(ii) the plurality of lens systems 520 c, 520 d form an arrayed lenssystem 520′ which provides isolated or independent optical paths for theemission beams 505 c, 505 d. In general, an arrayed lens system 520′ mayhave any number of individual lens systems 520. Further, the individuallens systems 520 in an arrayed lens system 520′ may be identical or maydiffer.

An example of a single lens system 520 is shown in FIG. 1I. As shown inthis particular example, the single lens system includes a plurality ofvarious optical elements 522 in a paired lens configuration. In general,the single lens system 520 may be provided with one or more of any ofvarious optical elements 522 in any of various optical pathconfigurations. Emission signals 505 a, 505 b, 505 c, etc. emanate frominterrogation element 150 (which may be provided, for example, within aparticle processing region 400). The single lens system 520 receives aplurality of emission signals 505 and as these emission signals travelthrough the single lens system 520 they may overlap or at leastpartially overlap. At the downstream end of the optical path, collectionsignals 515 a, 515 b, 515 c, etc. pass through a spatial filter 510which may be positioned in a light collection or detection plane.

An example of an arrayed lens system 520′ is schematically illustratedin FIG. 1J. In this embodiment, the three individual lens systems 520 a,520 b, 520 c are illustrated as being identical and arranged inparallel. Further, there is shown a one-to-one association betweenemission signals 505 a, 505 b, 505 c and lens systems 520 a, 520 b, 520c. In other embodiments, the individual lens systems need not beidentical, need not be arranged in parallel, and need not haveone-to-one correspondence between the emission signals and the lenssystems. Further, in this embodiment, the signal detection system 800 isillustrated as an array of detectors 820. This particular array ofdetectors 820 is shown as including three individual detectors 810 a,810 b, 810 c which are arranged in parallel and which have a one-to-oneassociation between emission signals 505 a, 505 b, 505 c, lens systems520 a, 520 b, 520 c and detector 810 a, 810 b, 810 c. In general, theindividual detectors need not be identical, need not be arranged inparallel, and need not have one-to-one correspondence between theemission signals and/or the lens systems with the detectors. As comparedto a single lens system, an arrayed lens system eliminates or reducesthe amount of overlap of the optical paths of the emission signals fromthe interrogation element to the detection plane. Other elements, suchas filters, for example, a spatial angular filter (not shown), may beprovided.

FIG. 1K schematically illustrates a system for mitigating opticalcrosstalk by spatially mapping signals 505 (or 515, 605, 805) onto asignal detection system 800. Specifically, an array of sensor elementsS_(A), S_(B), S_(C), etc. may be associated with an array ofinterrogation sites I_(A), I_(B), I_(C), etc. in interrogation element150 such that the spatial mapping of the array of sensor elements S_(A),S_(B), S_(C), etc. is scrambled with respect to the array ofinterrogation sites I_(A), I_(B), I_(C), etc. In other words, although aone-to-one mapping may exist between any individual particleinterrogation site I and a sensor element S, the mapping is scrambledsuch that signals from any two neighboring interrogation sites I aremore randomly dispersed among the sensor elements and are notnecessarily detected by any two adjacent sensor elements. FIG. 1Kprovides two example scrambled spatial mapping schemes for a lineararray of interrogation sites I and a linear array of sensor elements S.Referring to FIG. 1K(i), interrogation site I_(A) is mapped to sensorelement S_(C); interrogation site I_(B) is mapped to sensor elementS_(A); interrogation site I_(C) is mapped to sensor element S_(E); etc.Although linear arrays are shown in these two examples, two-dimensionalor three-dimensional arrays may be employed. Further, temporal mapping(i.e., a time dimension) may be employed to further isolate neighboringemission signals from neighboring sensors. Even further, spatial mappingmay employ non-scrambled or scrambled mapping with unused sensors (i.e.,unmapped sensors) surrounding and/or spatially separating the active,mapped sensors.

As shown in FIG. 1K, according to certain embodiments, spatiallyscrambled light mapping may be achieved by using an optical fiber bundle620 in a signal relay system 600 as part of the optical path between theinterrogation sites I_(A), I_(B), I_(C), etc. and the array of sensorelements S_(A), S_(B), S_(C), etc. For example, in FIG. 1K(ii), opticalfiber 622 _(A-A) maps interrogation site I_(A) to sensor element S_(A);optical fiber 622 _(B-D) maps interrogation site I_(B) to sensor elementS_(D); optical fiber 622 _(C-B) maps interrogation site I_(C) to sensorelement S_(B); etc. Light mapping may be achieved using optical fibersor other optical elements (including reflective, refractive,diffractive, etc.).

In FIG. 1L, schematic illustrations of an eight-by-eight optical sensorarray are shown. FIG. 1L(i) shows the spatial mapping scheme. Sixty-foursensors form a two-dimensional array and are numbered across the rows,starting at the bottom right with sensor number 1 and ending at the topleft with sensor number 64. The sensor number is shown in the top rightcorner of each sensor grid. Twelve interrogation sites (in thisembodiment, twelve microfluidic flow channels) have been spatiallymapped onto theses 64 sensors. Fluidic channels one through twelve areeach associated with five different spectral signals (for example, fourdifferent fluorescent signals and a side scatter signal). Each channeland each spectral signal has been mapped to a sensor for a total ofsixty mapped sensors (12 channels times 5 signals per channel equalssixty). In this embodiment, the four corner sensors (grids 1, 8, 57 and64) in the sensor array are unmapped. Each grid is correspondinglynumbered with the associated channel identifier (in the lower leftcorner of the grid) and the associated signal identifier (in the lowerright corner of the grid). Thus, for example, sensor number 2 is locatedin the lower row, second from the right and this sensor is mapped tochannel 6 and fluorescent signal 1. Sensor 40 is located in the far leftcolumn, fifth row from the bottom. This sensor is mapped to channel 11and to a side scatter (ss) signal. In FIG. 1L(i), each channel isassigned a color code so that the spatial mapping of each channel isvisually apparent. For example, channel 1 has been mapped to grids 41,50, 51, 58 and 59 in the upper right region of the sensor array andchannel 12 has been mapped to grids 6, 7, 14, 15 and 24 in the lowerleft region of the sensor array.

FIGS. 1L(ii), (iii) and (iv) show the signal levels detected for each ofthe sensors for three different events. In FIG. 1L(ii), a fluorescentsignal 1 for a particle in microfluidic channel 8 has been detected.Sensor 45, shown highlighted, has been mapped to channel 8 andfluorescent signal 1. As shown in FIG. 1L(ii), this sensor detects anintensity of 73914. Neighboring sensors have non-zero detector valuesranging from 145 to 1032. These non-zero detector values indicatespatial cross-talk occurring across neighboring sensors. However, sensorelements for the neighboring fluidic channels (7 and 9) for the samespectral signal (1) (i.e., sensor grids 62 and 22, respectively)register relatively low detector values (28 and 23, respectively). InFIG. 1L(iii), a fluorescent signal 1 for a particle in microfluidicchannel 9 has been detected. Sensor 22, shown highlighted, has beenmapped to channel 9 and fluorescent signal 1. As shown in FIG. 1L(iii),this sensor detects an intensity of 75958. Neighboring sensors havenon-zero detector values ranging from 165 to 1085. These non-zerodetector values indicate spatial cross-talk occurring across neighboringsensors. However, sensor elements for the neighboring fluidic channels(8 and 10) for the same spectral signal (1) (i.e., sensor grids 45 and46, respectively) register relatively low detector values (35 and 36,respectively). In FIG. 1L(iv), a fluorescent signal 1 for a particle inmicrofluidic channel 8 has been detected and a fluorescent signal 1 fora particle in microfluidic channel 9 has been simultaneously detected.Sensor 45 (channel 8, signal 1), shown highlighted, detects an intensityof 59917; sensor 22 (channel 9, signal 1), shown highlighted, detects anintensity of 64045. These detected intensity values are significantlygreater than values that would have been expected due to crosstalk alone(as determined from (ii) and (iii)), and thus, it can be reliablydetermined that simultaneous events occurred in these neighboringchannels. Further, the measured intensities are substantially free ofcrosstalk noise and the actual detected values may be relied upon.

According to another aspect, FIG. 1M schematically illustrates thatoptical crosstalk may be mitigated by spectrally mapping signals 505 (or515, 605, 805) onto the signal detection system 800. The signal pathsmay be scrambled or interwoven to minimize the amount of spectralcontent from adjacent interrogation sites I_(A), I_(B), etc. reachingadjacent (or near adjacent) sensor elements S_(A), S_(B), S_(C), etc.and/or to minimize any overlap in spectral signals from any given eventat an interrogation site I reaching adjacent (or near adjacent) sensorselements S_(A), S_(B), S_(C), etc. According to certain embodiments,scrambling may be achieved using optical filters or other spectralselection elements 730 to isolate the spectral signals 705. Opticalfibers 622 (or other optical elements) may be used to steer or directthe spectral signals 705 to specific sensors elements S_(A), S_(B),S_(C), etc. Focusing elements (not shown) may be provided between thespectrally mapped signals and the sensor elements.

For example, referring to FIG. 1M, a particle may be interrogated atsite I_(A) thereby emitting signal 505 a. This signal may be collectedand filtered according to its spectral content, e.g. according to itsfluorescent characteristics, into an array of spectral signals 705_(A1), 705 _(A2), 705 _(A3), 705 _(A4), etc. According to someembodiments, other signals such as a side scatter signal, a forwardscatter signal, an extinction signal, etc., associated with the sameparticle or interrogation event and may also be included in the array of“spectral” signals 705. Thus, in a general sense, an array of “spectral”signals may include any signal associated with a specific interrogationevent. The array of sensor elements S_(A), S_(B), S_(C), etc. may beassociated with the array of spectral signals 705 _(A1), 705 _(A2), 705_(A3), etc. on a one-to-one basis such that the spectral mapping of thearray of sensor elements S_(A), S_(B), S_(C), etc. is scrambled withrespect to the array of spectral signals 705 _(A1), 705 _(A2), 705_(A3), etc. In other words, although a one-to-one correspondence mayexist between any individual particle interrogation site I and a sensorelement S, the mapping is scrambled such that signals from any givenevent may be more randomly dispersed among the sensor elements. Further,the spectral mapping of the array of sensor elements S_(A), S_(B),S_(C), etc. may be scrambled with respect to the array of spectralsignals 705 _(A1), 705 _(A2), 705 _(A3), etc. and also with respect tothe array of interrogation sites I_(A), I_(B), I_(C), etc.

FIG. 1M provides an example scrambled spectral mapping scheme for alinear array of interrogation sites I, linear arrays of spectral signals705 and a linear array of sensor elements S. Thus, for example, aspectral signal 705 _(A1) associated with an event at interrogation siteI_(A) is mapped to sensor element S_(A); a spectral signal 705 _(A2)associated with the same event at interrogation site I_(A) is mapped tosensor element S_(C); spectral signal 705 _(A3) is mapped to sensorelement S_(E); etc. A spectral signal 705 _(B1) associated with an eventat interrogation site I_(B) is mapped to sensor element S_(G); aspectral signal 705 _(B2) associated with the same event atinterrogation site I_(B) is mapped to sensor element S_(E); spectralsignal 705 _(B3) is mapped to sensor element S_(D); etc. As with thespatial mapping described above, although linear arrays are shown inthese examples, two-dimensional or three-dimensional arrays may beemployed. An optical fiber bundle 620 in a signal relay system 600 mayprovide at least part of the optical path between the interrogationsites I_(A), I_(B), etc. and the array of sensor elements S_(A), S_(B),S_(C), etc. Optionally, mapping may be achieved using other opticalelements (including reflective, refractive, diffractive, etc.).

In FIG. 1N, schematic illustrations of an eight-by-eight optical sensorarray are shown similar to the illustrations of FIG. 1L. FIG. 1N(i)shows the spatial mapping scheme of twelve interrogation sites and fivespectral signals per site being mapped to sixty-four sensors provided ina two-dimensional array. For ease of explanation, in this embodiment thespecific mapping scheme is the same as that shown in FIG. 1L. In FIG.1N(i), each spectral signal is assigned a color code so that thespectral mapping is visually apparent. For example, spectral signals 1have been mapped to grids 2, 7, 19, 20, 22, 23, 42, 43, 45, 46, 59 and62 of the sensor array. Each of these mapped spectral signalscorresponds to a different interrogation site (e.g., a differentmicrofluidic channel). Spectral signals 3 have been mapped to grids 10,11, 13, 14, 27, 30, 34, 37, 51, 52, 54 and 55 of the sensor array.

FIGS. 1N(ii), (iii) and (iv) show the signal levels detected for each ofthe sensors for three different events. In FIG. 1N (ii), a fluorescentsignal 1 for a particle event in microfluidic channel 8 has beendetected by sensor 45, shown highlighted, as having an intensity of73914. Neighboring sensors have non-zero detector values ranging from145 to 1032. These non-zero detector values indicate cross-talkoccurring across neighboring sensors. However, sensor elements for thespectral signals (2, 3, 4, and ss) associated with the sameinterrogation site (channel 8) (i.e., sensor grids 39, 30, 29 and 32,respectively) register relatively low detector values (32, 53, 78 and 7,respectively). In FIG. 1N(iii), a fluorescent signal 2 for a particleevent in microfluidic channel 8 has been detected at sensor 39, shownhighlighted, as having an intensity of 69165. Neighboring sensors havenon-zero detector values ranging from 96 to 1091. These non-zerodetector values indicate cross-talk occurring across neighboringsensors. Sensor elements for the spectral signals (1, 3, 4, and ss)associated with the same interrogation site (channel 8) (i.e., sensorgrids 45, 30, 29 and 32, respectively) register relatively low detectorvalues (106, 209, 113 and 96, respectively). In FIG. 1N(iv), fluorescentsignals 1 and 2 have been simultaneously detected for a particle eventin microfluidic channel 8. Sensor 45 (channel 8, signal 1), shownhighlighted, detects an intensity of 64440; sensor 39 (channel 8, signal2), shown highlighted, detects an intensity of 59890. These detectedintensity values are significantly greater than values that would havebeen expected due to crosstalk alone (as determined from (ii) and(iii)), and thus, it can be reliably determined that the measuredintensities are substantially free of crosstalk noise and the actualdetected values may be relied upon.

FIG. 1O schematically illustrates a system for mitigating opticalcrosstalk by spatially and spectrally mapping signals 515 onto adetector array within a signal detection system 800. Specifically, anarray of sensor elements S_(A), S_(B), S_(C), etc. may be associatedwith an array of interrogation sites I_(A), I_(B), I_(C), etc. ininterrogation element 150 such that the spatial mapping of the array ofsensor elements S_(A), S_(B), S_(C), etc. is scrambled with respect tothe array of interrogation sites I_(A), I_(B), I_(C), etc. Further, thearray of sensor elements S_(A), S_(B), S_(C), etc. may be associatedwith the array of spectral signals 705 _(A1), 705 _(A2), 705 _(A3), etc.on a one-to-one basis such that the spectral mapping of the array ofsensor elements S_(A), S_(B), S_(C), etc. is scrambled with respect tothe array of spectral signals 705 _(A1), 705 _(A2), 705 _(A3), etc.Referring to FIG. 1O, a radiation source 200 may be spatially filter viamask 410 having apertures 412 a-412 f. Each aperture 412 is associatedwith an interrogation site IA-IE (e.g., a microfluidic channel) in thedetection plane 150. The apertures may be of the same or differentsizes. Further, in some embodiments a plurality of apertures may beassociated with an interrogation site. This plurality of apertures mayform a pattern that may assist in the evaluation of the emitted signalsand/or that may contain information about the interrogation site itself.

A single lens systems 520 collects emission signals 505 from theplurality of interrogation sites. Collection signals 515 may be focusedat focal plane 516. A spatial filter 710 having apertures 712 a-712 ecoincident with focal plane 516 allows signals 515 to be transmitted tofiber bundle 620. Fiber bundle 620 may spatially and/or spectrallyscramble and map signals A, B, C, etc. to sensors S_(A), S_(B), S_(C),etc.

Specific embodiments of the features described above will now bedescribed in conjunction with various particle processing systems 100 inaccordance with aspects presented herein.

FIG. 2 illustrates a specific embodiment of a portion of a particleprocessing system 100 that may be provided to minimize crosstalk. Inthis embodiment, the emission signal collection system 500 includes asingle lens system 520 provided as an optical column 550 with a spatialfilter 510 and/or a spectral filter. For this particular embodiment, thespatial filter 510 may be an image plane confocal aperture system.Signal conditioning system 700 includes a spectral filter, for example,a dichroic block array 720. Signal detection system 800 is provided as adetector array 820. Signal relay system 600 is provided as a spatiallyand/or spectrally scrambled bundle of fiber optics 620. As noted aboveand referring also to FIG. 1A, the signal relay system 600 may beprovided before or after the signal conditioning system 700.

FIG. 3A schematically illustrates a particle processing system 100 aaccording to another aspect. Similar to particle processing system 100illustrated in FIG. 1A, particle processing system 100 a may include aradiation source system 200, a radiation beam control system 300 a, aparticle processing region 400, an emission radiation signal collectionsystem 500, a signal relay system 600, a signal conditioning system 700(which may include a dichroic array), a signal detection system 800, andan electronics system 900. In this particular embodiment, radiationsource system 200 includes one or more radiation sources 210 that emit abeam of electromagnetic radiation 205. Beam 205 is transmitted to a setof beam shaping optics 220 via a plurality of minors 211. The shapedbeam 205 is then transmitted to a radiation beam control system 300 a.As with the embodiment described in FIG. 1A interrogation beam 215 isused to illuminate portions of the detection plane 150, which may beprovided within particle processing region 400. Emission signals 505 areemitted from the interrogation element 150 (e.g., due to particles beinginterrogated by the interrogation beam 215), collected by emissionsignal collection system 500 and transmitted to signal detection system800 via a signal relay system 600 and/or a signal conditioning system700.

Signal detection system 800 receives detector input signals 805 andconverts these into electrical signals 905 for transmission toelectronics 900. The signal detection system 800 may include a singlesensor (or detector) 810 or a plurality of sensors (or detectors). Forexample, a single detector may be provided one-to-one for each spectralsignal for each interrogation site (e.g., for each microfluidic channel,well, etc.); a single detector may be provided one-to-one for eachinterrogation site; a single detector may be provided for a plurality ofinterrogation sites; a single detector may be provided for each spectraldomain (i.e., color) across a plurality of interrogation site, etc.

In this particular embodiment, the radiation beam control system 300 amay include an optical scanner 320, a mirror 311, and a segmented mirror313. Optical scanner 320 is provided with an angular scanning capabilityfor scanning over an angular range a. An optical scanner may be providedby, for example, a galvanic mirror, an electro-optical scanner, anacousto-optical scanner, etc. A segmented mirror 313 suitable for use inthis embodiment has been described in U.S. Pat. No. 7,298,478 issuedAug. 18, 2009 to Gilbert et al., the contents of which are incorporatedby reference herein in their entirety. In other embodiments, other ordifferent optical elements may be included in the radiation beam controlsystem's optical path (e.g. free space optics such as refractive,reflective, diffractive, etc. elements and/or fiber optics and/orwaveguides).

During the course of a single scan, optical scanner 320 directsinterrogation beam 215 along interrogation beam path 215 a, then alonginterrogation beam path 215 b, then along interrogation beam path 215 c,etc. Each interrogation beam path 215 a, 215 b, 215 c, etc. isassociated with a segment of segmented minor 313, which in turn isassociated with an interrogation site in the detection plane 150. Thus,for example, when optical scanner 320 directs interrogation beam 215along interrogation beam path 215 a, particles within microfluidicchannel 422 a may be illuminated.

FIG. 3B illustrates how scan illumination may be used to reduce and/oraccount for optical crosstalk in the signal received by the signaldetection system 800 when a single detector is provided perinterrogation site per spectral domain (i.e., color). FIG. 3Billustrates idealized timing diagrams for scanning and detection ofparticles flowing within first and second microfluidic channels, whereinthe optical scanner 320 scans between the first and second channels.(Although shown for explanation purposes with respect to only twochannels in FIG. 3B, the particle processing system 100 a may generallyinclude any number of interrogation sites.) FIG. 3B(i) schematicallyillustrates a particle P flowing along a flow axis within either thefirst or the second microfluidic channel. In this embodiment, regions ofthe flow are configured to be illuminated via apertures 412. FIG. 3B(ii)illustrates a typical scan illumination pattern SI₁ (intensity versustime) as may be applied to a first microfluidic channel; FIG. 3B(iii)illustrates a typical scan illumination pattern SI₂ as may be applied toa second microfluidic channel. As the particle P flows within theaperture region 412 it is illuminated multiple times as the opticalscanner 320 directs the interrogation beam across the plurality ofchannels. Typically, the footprint (not shown) of the interrogation beam215 extends over the entire aperture region. The pulse delay between theinterrogation beam 215 scanning the first channel (see (ii)) and thenscanning the second channel (see (iii)) is shown as Δt.

FIG. 3B(iv) illustrates a typical detected signal DS₁ pattern as may bedetected by a first detector associated with a particle traveling alongthe first microfluidic channel (or residing within a first interrogationsite); FIG. 3B(v) illustrates a typical detected signal DS₂ pattern asmay be detected by a second detector associated with a particletraveling along the second microfluidic channel (or residing within asecond interrogation site). Specifically, FIG. 3B(iv) illustrates thatthe detector senses the detected signal DS₁ due to the particletraveling within the first channel and also may sense a crosstalk signalCS₂ due stray signals emanating from the second channel. However, due tothe pulse delay Δt experienced as the interrogation beam 215 scans thefirst channel and then scans the second channel, any crosstalk signalsCS₂ detected by the first detector that are associated with scanning ofthe second microfluidic channel (see (iii)) will be out-of-phase withthe signal emanating from the first microfluidic channel (see (ii)).Similarly, referring to FIG. 3B(v) any crosstalk signals CS₁ detected bythe second detector that are associated with scanning of the firstmicrofluidic channel (see (ii)) will be out-of-phase with the detectedsignal DS₂ emanating from second microfluidic channel (see (iii)). Thus,in-phase signals due to the detection of a particle within the scannedchannel may be isolated from the out-of-phase crosstalk signals due toadjacent channels being scanned.

In the above embodiment, the illumination or excitation apertures orpinholes 412 of the plurality of channels were assumed to be alignedwith one another along the longitudinal lengths of the channels. Inother words, as shown in FIG. 3C(i), the apertures 412 of a firstchannel 422 a are positioned side-by-side with the apertures 412 of thesecond channel 422 b. In another embodiment, referring to FIG. 3C(ii),the illumination or excitation apertures 412 need not be aligned, butmay be relatively staggered along the longitudinal lengths of thechannels 422 a, 422 b. A staggered aperture pattern may further reduceoptical crosstalk at the illumination stage.

FIG. 3D schematically illustrates another embodiment of particleprocessing system 100 a′ wherein the plurality of detectors 810 a, 810b, 810 c, etc. have been replaced with a single detector 810 d. Detector810 d is configured to receive signals from a plurality of interrogationsites 155 a, 155 b, 155 c, etc. for a single spectral or polarizationdomain (i.e., color).

FIG. 3E illustrates how scan illumination may be used to reduce and/oraccount for optical crosstalk in the signal received by the signaldetection system 800 when the single detector 810 d as shown in FIG. 3Dis configured to receive the signals from a plurality of interrogationsites 155 a, 155 b, 155 c, etc. for a single spectral domain (i.e.,color). For example, in the context of a particle processing region 400,FIG. 3E illustrates idealized timing diagrams for scanning and detectionof particles flowing within first and second microfluidic channels 422a, 422 b, wherein the optical scanner 320 scans between the first andsecond channels. As described above with respect to FIG. 3B(i), aparticle P may travel along a flow axis within either the first or thesecond microfluidic channel 422 a, 422 l and regions of the flow areconfigured to be illuminated via apertures 412. FIG. 3E(i) illustrates atypical scan illumination pattern SI₁ (intensity versus time) as may beapplied to a first microfluidic channel 422 a; FIG. 3E(ii) illustrates atypical scan illumination SI₂ pattern as may be applied to a secondmicrofluidic channel 422 b. FIG. 3E(iii) illustrates a typical detectedsignal DS₁ pattern as may be detected by the single detector for a firstparticle traveling along the first microfluidic channel 422 a and asecond particle simultaneously traveling along the second microfluidicchannel 422 b. The detected signals DS₁ due to the scanning pulsesinterrogating the first channel and the detected signals DS₂ due to thescanning pulses interrogating the second channel are alternativelyreceived (interleaved) by the detector 810 d. Given a known scanningpulse delay Δt, and referring to FIG. 3E(iv), these interleaved signalsmay be electronically separated (for example, using a demultiplexer 920)into electronic signals ES₁ that represent the event in the firstchannel and electronic signals ES₂ that represent the event in thesecond channel.

FIG. 4 schematically illustrates a particle processing system 100 baccording to another aspect. Specifically, FIG. 4 illustrates anembodiment of a particle processing system provided with a scan blockeracross the illumination source. Similar to particle processing system100 a illustrated in FIG. 3A, particle processing system 100 b mayinclude a radiation source system 200, a radiation beam control system300 b, a particle processing region 400, an emission signal collectionsystem 500, a signal relay system 600, a signal conditioning system 700,an signal detection system 800 and an electronics system 900. In thisparticular embodiment, the radiation beam control system 300 b includesan illumination beam spatial modulator 330 (rather than the opticalscanner 320 included in FIGS. 3A and 3D). Illumination beam spatialmodulator 330 may include a spatial filter 310 b. Spatial filter 310 ballows one or more portions of the radiation beam 205 to propagate tothe detection plane 150, while blocking the remainder of the radiationbeam 205. Those portions of the radiation beam 205 not blocked by thespatial filter 310 b are provided as interrogation beams 215 thatinterrogate select interrogation sites 155. Spatial filter 310 b may bea movable mask that is translated and/or rotated so that interrogationsites (or subsets of interrogation sites) are sequentially illuminatedby the portions of the radiation beam not blocked by the spatial filter.Alternatively, or additionally, the spatial filter 310 b may include oneor more shutters that open and close to sequentially open or closeapertures. As a non-limiting example, the spatial filter 310 b mayinclude a plurality of scanning slits, choppers, rotating chopperwheels, acousto-optic modulators (AOM), electro-optical modulators(EOM), etc. Other scanning spatial modulators as would be known topersons of ordinary skill in the art may be suitable. The segmentedmirror 313 of FIGS. 3A and 3D may be included in this embodiment.

At a first instance in time, illumination beam spatial modulator 330allows interrogation beam 215 a to interrogate channel 422 a, whileblocking beams 215 b, 215 c, etc. from illuminating the detection plane150. At a second instance in time, movable spatial filter 310 b has beentranslated and/or rotated so that interrogation beam 215 b is allowed tointerrogate channel 422 b, while beams 215 a, 215 c, etc. are blockedfrom illuminating the detection plane 150. Thus, each of theinterrogation sites 155 may be sequentially interrogated.

The idealized timing diagrams of FIG. 3B are applicable to the particleprocessing system 100 b of FIG. 4 when a single detector is provided permicrofluidic channel per spectral domain (i.e., color). Further, theidealized timing diagrams of FIG. 3E are applicable to the particleprocessing system 100 b of FIG. 4 as modified to include a singledetector configured to receive signals from a plurality of interrogationsites for a single spectral domain (i.e., color) (see for example, thesingle detector 810 d shown in FIG. 3D). FIGS. 3B and 3E illustrate howscanning across interrogation sites with an illumination beam spatialmodulator 330 may be used to reduce and/or account for optical crosstalkin the signal received by the signal detection system 800.

FIG. 5 schematically illustrates a particle processing system 100 caccording to yet another aspect. Similar to particle processing system100 a illustrated in FIG. 3A, particle processing system 100 c mayinclude a radiation source system 200 c, a radiation beam control system300 c, a particle processing region 400, an emission signal collectionsystem 500, a signal relay system 600, a signal conditioning system 700,an signal detection system 800 and an electronics system 900. In thisparticular embodiment, the radiation source system 200 c includes apulsed illumination array 230. Pulsed illumination array 230 includes aplurality of radiation sources 210 a, 210 b, 210 c, etc. that may bepulsed (i.e., turned on and turned off). The radiation sources 210 a,210 b, 210 c, etc. may be pulsed independently of one another or theymay be controlled with respect to each other or with respect to a commonbasis. Referring to FIG. 5, each radiation source 210 a, 210 b, 210 c,etc. may be associated with a single interrogation site 155 a, 155 b,155 c, etc. (as a non-limiting example, radiation source 210 a mayilluminate a detection region within a microfluidic channel 422 a;radiation source 210 b may illuminate a detection region within amicrofluidic channel 422 b; radiation source 210 c may illuminate adetection region within a microfluidic channel 422 c; etc.).Alternatively and/or additionally, radiation sources 210 a, 210 b, 210c, etc. may each be associated with a plurality of interrogation sites155. Even further, each interrogation site 155 may be associated with asingle radiation source 210 or with more than one radiation source 210.

Further, in this particular embodiment, the radiation beam controlsystem 300 c includes a pulse generator 340 (rather than the opticalscanner 320 included in FIGS. 3A and 3D or the illumination beam spatialmodulator 330 of FIG. 4). Electronics system 900 may control the pulsegenerator 340, which in turn controls the illumination of theinterrogation sites by the pulsed radiation sources 210 a, 210 b, 210 c,etc. of the pulsed illumination array 230. The pulse generator 340 maydelay or stagger the pulses being generated. As a non-limiting example,a pulsed source delay timing array may be provided as [(t-t₀); (t-t₁);(t-t₂); etc.].

For example, the pulse delay generator 340 may control the pulseactivation widths (i.e., the time over which an interrogation beam 215is produced by the radiation source 210) of each of the radiationsources. According to one embodiment, each of the pulsed radiationsources may have identical pulse activation widths. According to anotherembodiment, the pulse widths need not be identical, but rather, they maydiffer across the plurality of radiation sources. Further, the pulsewidths for any given radiation source may remain constant or they mayvary.

Additionally and/or alternatively, the pulse generator 340 may controlthe delay time (i.e., the time between the pulse activation “on” signalsbeing sequentially sent to the radiation sources 210 a, 210 b, 210 c,etc.). A delay time between sequentially activated pulses may be equalto the pulse activation width, in which case the pulse of the firstradiation source 210 a will end at the same time that the pulse of thesecond radiation source 210 b begins. Optionally, a delay time betweensequentially activated pulses may be greater than the pulse activationwidths, in which case there will be a break or a gap between when afirst radiation source 210 a has ended its pulse and when the secondradiation source 210 b has begun its pulse. On the other hand, a delaytime between sequentially activated pulses may be less than the pulseactivation widths, in which case there will be an overlap between when afirst radiation source 210 a has ended its pulse and when the secondradiation source 210 b has begun its pulse. The delay time forsequentially activating pulses may be constant, may differ and/or mayvary.

According to an example embodiment, at a first instance in time t_(o),radiation source 210 a of pulse generator 340 is activated such thatinterrogation beam 215 a illuminates channel 422 a. While radiationsource 210 a is activated, radiation sources 210 b, 210 c, etc. remainoff. At a second instance in time t₁, radiation source 210 a isde-activated or turned off. At a third instance in time t₂, radiationsource 210 b of pulse generator 340 is activated. Radiation source 210 bof pulse generator 340 may generate an interrogation beam 215 b thatilluminates channel 422 b. At a fourth instance in time t₃, radiationsource 210 b is de-activated or turned off.

According to certain embodiments, radiation sources 210 a, 210 b, 210 c,etc. may be sequentially spatially arranged within pulsed illuminationarray 230 and excitation 210 a, 210 b, 210 c, etc. may be sequentiallypulsed. According to other embodiments, radiation sources 210 a, 210 b,210 c, etc. may be sequentially spatially arranged within pulsedillumination array 230 and radiation sources 210 a, 210 b, 210 c, etc.may be pulsed out-of-sequence (i.e., first 210 a may be pulsed, then 210f may be pulsed, then 210 b may be pulsed, then 210 e may be pulsed, andso on). Thus, each of the interrogation sites 155 may be sequentiallyinterrogated or, in alternative embodiments, each of the interrogationsites 155 may be more randomly illuminated or pulsed.

The idealized timing diagrams of FIG. 3B are equally applicable to theparticle processing system 100 c of FIG. 5 when a single detector isprovided per microfluidic channel per spectral domain (i.e., color) andwhen pulsed illumination as described above (rather than scannedillumination) is applied to the interrogation sites 155. Further, theidealized timing diagrams of FIG. 3E are applicable to the particleprocessing system 100 c of FIG. 5 as modified to include a singledetector configured to receive signals from a plurality of interrogationsites 155 for a single spectral domain (i.e., color) (see for example,the single detector 810 d shown in FIG. 3D) and when pulsed illuminationas described above (rather than scanned illumination) is applied to theinterrogation sites. Thus, FIGS. 3B and 3E also illustrate how,according to certain embodiments, pulsed interrogation of a plurality ofinterrogation sites 155 with a pulsed illumination array 230 and a pulsegenerator 340 may be used to reduce and/or account for crosstalk in thesignals received by the signal detection system 800.

FIG. 6A and FIG. 6B show two embodiments of a particle processing system100 that may utilize pulse discrimination with a speed of light delay.For example, in FIG. 6A a particle processing system 100 d includes aradiation beam control system 300 d having an array 360 of radiationbeam transmission channels C₁, C₂, C₃, etc. Each of these transmissionchannels C₁, C₂, C₃, etc. may have differing indices of refraction (n₁,n₂, n₃, etc.). These differing refraction indexes result in theradiation beam 205 being transmitted at differing speeds of light, thusdeveloping a delay in the timed delivery of the interrogation beams 215a, 215 b, 215 c, etc. between the channels. When a pulsed light source250 is triggered (for example, via a pulse synchronization signal PSSprovided by electronics system 900), a plurality of time-staggeredpulsed radiation beams 215 a, 215 b, 215 c may be produced. As onenon-limiting example, the pulsed light source 250 may be provided as apulsed or quasi-continuous-wave laser.

As another example, in FIG. 6B a particle processing system 100 eincludes a radiation beam control system 300 e provided with an array370 of fiber optic transmission channels F₁, F₂, F₃, etc. havingdifferent lengths (l₁, l₂, l₃, etc.). The differing lengths of theoptical fibers result in differing times for transmitting theinterrogation beams 215, thus, again developing a timed delay of theinterrogation beams 215 to the interrogation sites. In general, anoptical delay may be developed by directing the interrogation beams 215along different paths having different lengths and/or differenttransmissivities. Thus, as with particle processing system 100 d, when apulsed light source 250 is triggered in particle processing system 100e, a plurality of time-staggered pulsed radiation beams 215 a, 215 b,215 c may be produced.

The idealized timing diagrams of FIG. 3B are also applicable to theparticle processing system 100 d of FIG. 6A (and also to the particleprocessing system 100 e of FIG. 6B) when a single detector is providedper microfluidic channel per spectral domain (i.e., color) and whenpulsed illumination as described above (rather than scannedillumination) is applied to the interrogation sites 155. Further, theidealized timing diagrams of FIG. 3E are applicable to the particleprocessing system 100 d of FIG. 6A (and also to the particle processingsystem 100 e of FIG. 6B) as modified to include a single detectorconfigured to receive signals from a plurality of interrogation sites155 for a single spectral domain (i.e., color) (see for example, thesingle detector 810 d shown in FIG. 3D) and when pulsed illumination asdescribed above (rather than scanned illumination) is applied to theinterrogation sites 155. Thus, FIGS. 3B and 3E also illustrate how,according to certain embodiments, pulsed interrogation of a plurality ofinterrogation sites 155 using a speed of light delay to discriminate thepulses as embodied, for example, in the array 360 of refractive channels(FIG. 6A) or in the array 370 of optical fibers (FIG. 6B), may be usedto reduce and/or account for optical crosstalk in the signals receivedby the signal detection system 800.

Referring now back to FIG. 5, a variation 100 c′ of particle processingsystem 100 c is described. In the embodiment 100 c discussed above,pulsed interrogation beams 215 a, 215 b, 215 c, etc. are directed to theinterrogation sites 155 a, 155 b, 155 c, etc. of the detection plane150. The radiation source system 200 c may include a pulsed illuminationarray 230 which includes a plurality of radiation sources 210 a, 210 b,210 c, etc. that may be pulsed (i.e., turned on and turned off) eitherindependently or in a coordinated manner. A pulse generator 340 may beused to control the illumination of the interrogation sites bycontrolling the pulsed radiation sources 210 a, 210 b, 210 c, etc. ofthe pulsed illumination array 230. In a variation, the signal detectionsystem 800′ of particle processing system 100 c′ includes one or morefluorescence lifetime detectors 830. Detectors 830 measure the lifetimeand/or the decay characteristics of the fluorophore signal (in additionto and/or alternatively to measuring the signal's intensity). Thesesignals may be differentiated based on differences in the exponentialdecay rate of the emitted fluorescence. As such, signals havingdifferent fluorescence decay rates may be differentiated, even if thewavelengths of the signals are the same. When fluorescence lifetimes arebeing detected, ultrashort excitation pulses (such as may be provided,for example, by a pulsed or quasi-continuous-wave laser) may bepreferable.

Similarly, referring back to FIGS. 6A and 6B, respective variations 100d′, 100 e′ of the particle processing systems 100 d and 100 e may beprovided with a signal detection system 800′ that includes one or morefluorescence lifetime detectors 830.

FIG. 7A (similar to FIG. 3B when discussed in the context of FIG. 5 andFIGS. 6A and 6B) illustrates how pulsed illumination may be used toreduce and/or account for optical crosstalk in the signal received bythe signal detection system 800′ when a single fluorescence lifetimedetector 830 is provided per interrogation site per spectral domain(i.e., color). FIG. 7B (similar to FIG. 3E when discussed in the contextof FIG. 5 and FIGS. 6A and 6B) illustrates how pulsed illumination maybe used to reduce and/or account for crosstalk in the signal received bythe signal detection system 800′ when a single fluorescence lifetimedetector 830 is configured to receive the signals from a plurality ofinterrogation sites for a single spectral domain (i.e., color). As shownin FIGS. 7A and 7B, the pulsed illumination pattern may be the same asshown in FIGS. 3B(ii) and (iii) and in FIGS. 3E(i) and 3E(ii). However,FIG. 7A(iv) illustrates a typical detected signal DS₁′ pattern as may bedetected by a first fluorescence lifetime detector 830 associated with aparticle or other sample within a first interrogation site 155 a; FIG.7A(v) illustrates a typical detected signal DS₂′ pattern as may bedetected by a second fluorescence lifetime detector 830 associated witha particle or other sample within a second interrogation site 155 b.Specifically, FIG. 7A(iv) illustrates that the fluorescence lifetimedetector 830 senses the detected signal DS₁′ due to the particle withinthe first interrogation site and also may sense a crosstalk signal CS₂′due to stray signals emanating from the second interrogation site.However, due to the pulse delay Δt experienced as the interrogation beam215 a pulses the first interrogation site and then the interrogationbeam 215 b pulses the second interrogation site, any crosstalk signalsCS₂′ detected by the first fluorescence lifetime detector 830 that areassociated with pulsing of the second interrogation site (see (iii))will be out-of-phase with the signal emanating from the firstinterrogation site (see (ii)). Similarly, referring to FIG. 7A(v) anycrosstalk signals CS₁′ detected by the second fluorescence lifetimedetector 830 that are associated with the first interrogation site (see(ii)) will be out-of-phase with the detected signal DS₂′ emanating fromsecond interrogation site (see (iii)). Thus, in-phase signals due to thedetection of a particle within the interrogation site may be isolatedfrom the out-of-phase crosstalk signals due to adjacent interrogationsites being pulsed.

FIG. 7B illustrates a typical detected signal DS₁′ pattern as may bedetected with a single fluorescence lifetime detector 830 for a firstparticle within a first interrogation site 155 a and a second particlesimultaneously within a second interrogation site 155 b. The detectedsignals DS₁′ due to the pulses interrogating the first interrogationsite 155 a and the detected signals DS₂′ due to the pulses interrogatingthe second interrogation site 155 b are alternatively received(interleaved) by the fluorescence lifetime detector 830. Given a knownscanning pulse delay Δt, and referring to FIG. 7B (iv), theseinterleaved signals may be electronically separated (for example, usinga demultiplexer 920) into electronic signals ES₁′ that represent theevent at the first interrogation site 155 a and electronic signals ES₂′that represent the event at the second interrogation site 155 b.Electronic signals ES₁′ are 90 degrees out-of-phase with electronicsignals ES₂′ in this embodiment.

FIGS. 8A and 8B schematically illustrate particle processing systems 100f and 100 f′ wherein the signal detection system 800 is synchronouslylocked to a radiation source system 200. Further, the synchronousmodulation may be controlled by a trigger associated with a signal fromthe detection plane 150.

FIG. 8A illustrates a radiation source system 200 having a plurality ofradiation sources 210 a, 210 b, 210 c, etc. for illuminating individualinterrogation sites 155. These radiation sources 210 a, 210 b, 210 c,etc. are triggered by a modulation controller 930. In this embodiment,the modulation controller 930 may independently trigger each of theradiation sources 210 a, 210 b, 210 c, etc. according to one or morepulsed time delays Δt₀, Δt₁, Δt₂, etc. The modulation controller 930also controls the signal detection system 800 so that signals detectedby the signal detection system 800 are synced to the interrogation beams215 illuminating the interrogation sites 155. In the embodiment of FIG.8A, the signal detection system 800 includes an amplifier array 840 andthe modulation controller 930 is synced thereto. Signals (e.g,extinction, scatter, etc.) from the detection plane 150 may also bedetected by a secondary detector 810 b. As one non-limiting example,light scatter 505′ from the interrogation sites 155 may be detected byone or more secondary detectors 810 b. These signals may be processed byelectronics system 900′ and provided to modulation controller 930.

FIG. 8B illustrates how locked-in detection may be used to reduce and/oraccount for crosstalk in the signal received by the signal detectionsystem 800 when a plurality of modulated radiation sources 210 a, 210 b,etc. is provided for a plurality of interrogation sites 155 according tothe embodiment of FIG. 8A. FIG. 8B illustrates idealized timing diagramsfor scanning and detection of particles flowing within first and secondmicrofluidic channels, wherein the modulation controller 930 modulatesthe radiation beam 215 at a single frequency f₁ for all interrogationsites (for example, two side-by-side microfluidic flow channels), butwith a pulse time delay triggering the illumination for each of theplurality of radiation sources. FIG. 8B(i) illustrates a typicalmodulated illumination pattern MI₁ (intensity versus time) as may beapplied to a first microfluidic channel; FIG. 8B(ii) illustrates atypical modulated illumination pattern MI₂ as may be applied to a secondmicrofluidic channel. As a particle P flows within the channel it wouldbe illuminated multiple times as the radiation sources 210 a, 210 b, 210c, etc. (and thus also the interrogation beams 215) are modulated. Thetrigger delay between an interrogation beam 215 illuminating the firstchannel and then an interrogation beam 215 illuminating the secondchannel is shown as Δt′.

FIG. 8B(iii) illustrates a typical detected signal DS₁ pattern as may bedetected by a first detector associated with a particle traveling alongthe first channel (or residing within a first interrogation site); FIG.8B(iv) illustrates a typical detected signal DS₂ pattern as may bedetected by a second detector associated with a particle traveling alongthe second channel (or residing within a second interrogation site).Specifically, FIG. 8B(iii) illustrates that the detector senses thedetected signal DS₁ due to the particle traveling within the firstchannel and also may sense a crosstalk signal CS₂ due stray signalsemanating from the second channel. However, due to the trigger delay Δtexperienced as the interrogation beam 215 illuminates the first channeland then the second channel, any crosstalk signals CS₂ detected by thefirst detector that are associated with illuminating the secondmicrofluidic channel (see (ii)) will be out-of-phase with the signalemanating from the first microfluidic channel (see (iii)). Similarly,referring to FIG. 8B(iv) any crosstalk signals CS_(I) detected by thesecond detector that are associated with illuminating the firstmicrofluidic channel (see (i)) will be out-of-phase with the detectedsignal DS₂ emanating from second microfluidic channel (see (iv)). Thus,in-phase signals due to the detection of a particle within theilluminated channel may be isolated from the out-of-phase crosstalksignals due to adjacent channels being illuminated.

FIG. 8C illustrates a particle processing system 100 f′ that differsfrom 100 f in that the plurality of radiation sources 210 a, 210 b, 210c, etc. are triggered by a modulation controller 930′. In thisembodiment, the modulation controller 930′ may independently triggereach of the radiation sources 210 a, 210 b, 210 c, etc. at one or morefrequencies f₁, f₂, f₃, etc. or with a varying frequency pattern f(t).

FIG. 8D illustrates how locked-in detection may be used to reduce and/oraccount for crosstalk in the signal received by the signal detectionsystem 800 when a plurality of modulated radiation sources 210 a, 210 b,etc. is provided according to FIG. 8C. FIG. 8D(i) illustrates a typicalmodulated illumination pattern MI₁ (intensity versus time) at a firstfrequency f₁ as may be applied to a first channel; FIG. 8D(ii)illustrates a typical modulated illumination MI₂ pattern at a secondfrequency f₂ as may be applied to a second channel. FIG. 8D(iii)illustrates a typical detected signal DS₁ pattern as may be detected bythe single detector for a first particle traveling along the firstchannel and a second particle simultaneously traveling along the secondchannel. The detected signals DS₁ due to the modulated pulsesinterrogating the first channel overlap the detected signals DS₂ due tothe modulated pulses interrogating the second channel. Given the knownfrequencies of the modulated pulses for each channel, these overlappedsignals may be electronically spectrally separated and/or mathematicallydeconvolved.

FIG. 9A schematically illustrates particle processing system 100 gwherein the signal detection system 800 identifies and isolates specificinterrogation sites via modulation filters. Thus, a spatial filter array440 may be provided as spatial illumination patterns and/or as spatialdetection patterns. These spatial illumination and/or detection patternsmay be provided by masks, aperture arrays, etc. In certain embodiments,patterned gray scale optical masks may be used. The patterned masks maybe associated one-to-one with the interrogation sites 155 (e.g., one ofthe microfluidic flow channels 422). Further, each interrogation site155 may be provided with a unique spatial filter pattern in which thefrequency, period, duty cycle, shape, and/or sized of the aperture inthe array may be varied.

According to another aspect, spectral illumination and/or detectionpatterns may be provided. As a non-limiting example, a spectral filterarray (e.g., a spectral optical mask) may be provided, wherein thetransmission spectrum of the pattern varies across the array. Thus, eachmicrofluidic flow channel 422 may have a distinct spectral pattern orsignature. According to other embodiments (not shown), any combinationof spatial modulation and spectral modulation may be combined. Thespatial and/or spectral illumination patterns may be provided in theillumination (object) plane, the detection image plane, etc.

Thus, referring to FIG. 9B(i) a first gray scale mask 440 a is shownposition over a first flow channel 422 a with a particle P1 flowingtherein and in FIG. 9B(ii) a second gray scale mask 440 b is shownposition over a second flow channel 422 b with a particle P2 flowingtherein. The gray scale bars of the first mask 440 a are wider andspacer farther apart than the gray scale bars of the second mask 440 b,and thus, the signals produced when the particle passes across the maskshave different pulse frequencies. In addition, the spatial frequency mayvary across the pattern for a given channel. The different frequenciesand varying intensity patterns allow these wavelets ψ₁(t), ψ₂(t) to beidentified, isolated and deconvolved (e.g., wavelet analysis) shouldcrosstalk from a neighboring channel be detected along with the detectedsignal from the illuminated channel. FIG. 9A illustrates a radiationsource system 200 having a single radiation source 210 for illuminatinga plurality of interrogation sites 155. By way of non-limiting example,the radiation source 210 may be a continuous wave laser. The radiationsource system 200 may additionally include beam shaping optics 220 (asshown). The signal detection system 800 may include a single detectorassociated one-to-one with an interrogation site or microfluidic flowchannel. Alternatively, the signal detection system 800 may include asingle detector associated with a plurality of interrogation sites ormicrofluidic flow channels.

Optionally, secondary signals 505′ from the detection plane 150 may bedetected by a secondary detector 810 b. As one non-limiting example,when fluorescence is the primary signal detected, light scatter from theinterrogation sites 155 may be detected by one or more secondarydetectors 810 b. As another example, an extinction signal may bedetected by a secondary detector 810 b. These secondary signals may beprocessed by electronics system 900 and may provide a trigger to thecontroller to illuminate or detect only in the presence of a particle.

FIG. 9C(i) shows wavelet illumination signals WIS₁, WIS₂ for first andsecond microfluidic flow channels 422 a, 422 b, respectively, when eachmicrofluidic flow channels is associated with a single detector. FIG.9C(ii) shows wavelet detected signals WDS₁, WDS₂ for first and secondmicrofluidic flow channels 422 a, 422 b, respectively, along withwavelet crosstalk signals WCS₂, WCS₁ for second and first microfluidicflow channels 422 b, 422 a, respectively, that have infiltrated theprimary detected signal. FIG. 9C(iii) the wavelet electronic signalsWES₁, WES₂ for first and second microfluidic flow channels 422 a, 422 b,respectively, the detected signals have been processed to eliminate thecrosstalk signal. FIG. 9D shows the process when a single detectorreceives signals from a plurality of microfluidic flow channels, as hasbeen describe in the context of scan illumination with respect to FIG.3E.

The particle processing system 100 h of FIG. 10 is very similar to theparticle processing systems 100 c, 100 c′ of FIG. 5. In FIG. 10, aspatial filter 640 which may selectively block (or selectively transmit)a signal from one or more interrogation sites 155 is supplied in thedetection path. Spatial filter 640 is linked to the pulse generator 340.As discussed with respect to particle processing system 100 c, pulsegenerator 340 controls the illumination of the individual interrogationsites 155 by the radiation sources 210 a, 210 b, 210 c, etc. of thepulsed excitation array 230. In the embodiment of particle processingsystem 100 h, pulse delay generator 340 also controls the opening andclosing (i.e., the selective transmission and the selective blocking) ofapertures associated with the signals from the individual interrogationsites 155. Thus, spatial filter 640 may be configured as a scanningdetection signal aperture blocker (or alternatively, as a scanningdetection signal aperture transmitter). When interrogation site 155 a isilluminated by interrogation beam 215 a, the pulse generator 340instructs spatial filter 640 to open the aperture associated with thedetection signal from interrogation site 155 a so that this signal maybe detected by detector 810. At the same time, the pulse generator 340may instruct spatial filter 640 to block some or all of any othersignals emanating from the detection plane 150. Similarly, wheninterrogation site 155 b is illuminated by interrogation beam 215 b, thepulse generator 340 instructs spatial filter 640 to open the apertureassociated with the detection signal from interrogation site 155 b sothat this signal may be detected by detector 810. At the same time, thepulse generator 340 may instruct spatial filter 640 to block some or allof any other signals emanating from the detection plane 150.

Thus, the idealized timing diagrams of FIG. 3B are also applicable tothe particle processing system 100 h of FIG. 10 when a single detectoris provided per microfluidic channel per spectral domain (i.e., color)and when detection signals from interrogation sites 155 a, 155 b, 155 c,etc. are selectively and synchronously transmitted. Further, theidealized timing diagrams of FIG. 3E are applicable to the particleprocessing system 100 h of FIG. 10 as modified to include a singledetector configured to receive signals from a plurality of interrogationsites 155 for a single spectral domain (i.e., color) (see for example,the single detector 810 d shown in FIG. 3D) and when detection signalsfrom interrogation sites 155 a, 155 b, 155 c, etc. are selectively andsynchronously transmitted. Thus, FIGS. 3B and 3E also illustrate how,according to certain embodiments, pulsed interrogation of a plurality ofinterrogation sites 155 coupled with selective and synchronoustransmission and/or blocking of individual interrogation sites 155 a,155 b, etc. may be used to reduce and/or account for crosstalk in thesignals received by the signal detection system 800.

FIGS. 11A and 11B schematically illustrate particle processing systems100 i and 100 i′ wherein the signals 905 a, 905 b, 905 c etc. sent fromthe signal detection system 800 to the electronics system 900 aresynchronized with pulsed interrogation beams 215 a, 215 b, 215 c, etc.In FIG. 11A, a pulse generator 340 controls the triggering pulsessupplied to the radiation sources 210 a, 210 b, 201 c, etc. and alsocontrols pulses that switch that activates the detection electronics900. Thus, for example, a pulse may be supplied to radiation source 210a via line t_(o) and at the same time a pulse may be supplied todetection switch 845 via line t₀′ so that the signal 905 a received bythe electronics system 900 is synchronized with the signal 505 emanatingfrom interrogation site 155 a. In FIG. 11B, a pulse generator 340controls the triggering pulses supplied to the radiation sources 210 a,210 b, 201 c, etc. and also triggers a detector gain/power pulseselector switch 345. The detector gain/power pulse selector switch 345selectively controls the gain/power associated with the individualdetectors 810. Thus, for example, a pulse may be supplied to radiationsource 210 a via line t_(o) and at the same time a pulse may be suppliedto gain/power pulse selector switch 345 so that signal 905 a may besynchronously received by the electronics system 900.

The idealized timing diagrams of FIG. 3B are applicable to the particleprocessing systems 100 i and 100 i′ of FIGS. 11A and 11B when a singledetector is provided per microfluidic channel per spectral domain (i.e.,color). FIG. 3B illustrates how synchronously switching detectionelectronics with the pulsed interrogation beams 215 supplied to theinterrogation sites may be used to reduce and/or account for crosstalk.

FIGS. 12A-12B schematically illustrate multiple embodiments of particleprocessing systems 100 k, 100 k′ that use heterodyne optical detectionsystems. As shown in FIG. 12A, for spatially coherent detectedradiation, the radiation source system 200 of particle processingsystems 100 k, may be, for example, an illumination array 270 driven ata plurality of frequencies (f_(sig1), f_(sig2), f_(sig3), . . .f_(sig n)) supplied by a first electronic frequency generator 954 a. Thefrequency driven illumination array 270 provides pulsed interrogationbeams 215 a, 215 b, 215 c, etc. to a plurality of interrogation sites155. A second electronic frequency generator 954 b provides thereference signals to drive an optical local oscillator 970 at areference frequency (f_(ref)) different than those reference signalsused to generate the interrogation beams 215. The reference frequency(f_(ref)) may preferably be greater than the frequencies used togenerate the interrogation beams 215. Emitted signals 505/805 and thereference signal from the optical local oscillator 970 are mixed anddetected by the detector array 800 at frequencies (f_(mixed1),f_(mixed2), f_(mixed3), . . . f_(mixed n)). Thus, each interrogationsite has an illumination/detection signal frequency f_(sig) associatedwith it; all interrogation sites share the same reference frequencyf_(ref); and each interrogation site also has a mixed frequencyf_(mixed) associated with it. The mixed frequency signal consists ofseveral harmonic combinations of the reference, illumination/detection,and crosstalk signals. The electronics system 900 demodulates theillumination/detection signal and the crosstalk signal from thereference frequency. Electronic filters then remove the unwantedcrosstalk frequency components from the illumination/detection signal ofeach channel.

FIG. 12B illustrates a particle processing system 100 k′ with a balancedoptical heterodyne detection system. Although similar to the particleprocessing system 100 k, in the balanced heterodyne system, theillumination/detection signals are mixed with the local oscillator on asecondary detector array. The signals from the secondary detector array800 c are subtracted from the primary detector array 800 a. Thisconfiguration enables noise generated by the local oscillator to besubtracted from the mixed illumination/detection signal.

FIG. 13 schematically illustrates an embodiment of a particle processingsystem 100 l that uses a heterodyne optical detection system ofspatially incoherent light. Thus, for this embodiment, the radiationsource system 200 may be, for example, a illumination array 270 drivenat a plurality of frequencies (f_(sig1), f_(sig2), f_(sig3), . . .f_(sig n)) supplied by an electronic signal generator 956. Theillumination array 270 provides modulated interrogation beams 215 a, 215b, 215 c, etc. to a plurality of interrogation sites 155. An electronicoscillator generator 954 c provides the reference signals at frequency(f_(ref)) which is greater than the frequency of the interrogation beams215. Signals 805 from the detector array 800 and signals from theelectronic oscillator generator 954 c are combined in electronics system900. As with the particle processing systems 100 k, 100 k′, eachinterrogation site has an illumination/detected signal frequency f_(sig)associated with it; all interrogation sites share the same referencefrequency f_(ref); and each interrogation site also have a mixedfrequency f_(mixed) associated with it. The mixed frequency signalconsists of several harmonic combinations of the reference,illumination/detection, and crosstalk signals. The electronics system900 demodulates the illumination/detection signal and the crosstalksignal from the reference frequency. Electronic filters then remove theunwanted crosstalk frequency components from the illumination/detectionsignal of each channel.

According to a variation (not shown) of the particle processing system100 l, a system using a homodyne optical detection system may beprovided. Homodyne detection uses a reference frequency to detectfrequency-modulated radiation. The reference signal may be supplied by alocal frequency generator. The signal and the local oscillator aresuperimposed at a mixer. In homodyne detection, the local frequencygenerator has the same frequency as the signal being detected (and,typically, they are both derived from the same source). Homodynedetection systems are generally insensitive to fluctuations in thefrequency of the source.

Although the systems, assemblies and methods of the present disclosurehave been described with reference to exemplary embodiments thereof, thepresent disclosure is not limited to such exemplary embodiments and/orimplementations. Rather, the systems, assemblies and methods of thepresent disclosure are susceptible to many implementations andapplications, as will be readily apparent to persons skilled in the artfrom the disclosure hereof. The present disclosure expressly encompassessuch modifications, enhancements and/or variations of the disclosedembodiments. Since many changes could be made in the above constructionand many widely different embodiments of this disclosure could be madewithout departing from the scope thereof, it is intended that all mattercontained in the drawings and specification shall be interpreted asillustrative and not in a limiting sense. Additional modifications,changes, and substitutions are intended in the foregoing disclosure.Accordingly, it is appropriate that the appended claims be construedbroadly and in a manner consistent with the scope of the disclosure.

1. A particle processing system comprising: a particle processingregion; and a signal processing system in communication with theparticle processing region; wherein the signal processing system isconfigured to reduce optical crosstalk between a plurality of opticalpaths to improve performance of the particle processing system.
 2. Theparticle processing system of claim 1, wherein the signal processingsystem includes: an optical system having optical signals emitted from aplurality of particle interrogation sites mapped via the plurality ofoptical paths to a plurality of sensor locations; wherein the mappingdefines an arrangement of the plurality of optical paths with at leastsome of the signals from adjacent particle interrogation sites beingdirected to sensor locations that are not adjacent.
 3. The particleprocessing system of claim 2, wherein the plurality of optical pathsincludes optical fibers or a fiber bundle.
 4. The particle processingsystem of claim 2, wherein the plurality of optical paths includesmirrors or steering elements.
 5. The particle processing system of claim2, wherein the optical system includes a large span optical system forcollection of the emitted optical signals from the plurality of particleinterrogation sites.
 6. A method of using particle processing system ofclaim 2, the method comprising the following steps: using a large spanoptical system for collection of the emitted optical signals from theplurality of particle interrogation sites; using one or more spatialfilters; using isolated optical pick-up systems; and using scrambledlight mapping, which may be spatial or spectral in nature.
 7. Theparticle processing system of claim 2, further comprising: amulti-element photo-multiplier tube array; wherein the multi-elementphoto-multiplier tube provides the plurality of sensor locations.
 8. Theparticle processing system of claim 7, further comprising: a dichroicblock, a detector, and a scrambled fiber bundle disposed between thedichroic block and the detector; image plane confocal apertures; asingle lens system; and a microfluidic chip array with illuminationapertures.
 9. The particle processing system of claim 2, wherein theplurality of optical paths includes a scrambled fiber bundlestrategically mapped to a sensor scheme that minimizes opticalcrosstalk.
 10. A method for minimizing optical crosstalk using theparticle processing system of claim 1, the method comprising: providingthe particle processing system, wherein the particle processing systemis configured to emit optical signals from a plurality of particleinterrogation sites; mapping the optical signals emitted from theplurality of particle interrogation sites to a plurality of sensorlocations; wherein the mapping defines and arrangement of the pluralityof optical paths with optical signals from at least some of the adjacentparticle interrogation sites being mapped to sensor locations that arenot adjacent.
 11. A method for minimizing optical crosstalk using theparticle processing system of claim 1, the method comprising: providingthe particle processing system, wherein the particle processing systemis configured to emit optical signals having a plurality of spectralwavelengths; mapping the optical signals to a plurality of sensorlocations; wherein the mapping defines an arrangement of the pluralityof optical paths with optical signals having common spectral wavelengthsbeing mapped to sensor locations that are not adjacent.
 12. A particleprocessing system comprising: a radiation source system configured tooptically interrogate a plurality of interrogation sites; a signaldetection system including a detector configured to receive opticalsignals emitted from the plurality of interrogation sites; wherein theradiation source system is configured to at least one of: (i)sequentially interrogate each of the plurality of interrogation siteswith a predetermined timed delay or (ii) simultaneously interrogate afirst interrogation region with a first predetermined signal frequencyand a second interrogation region with a second predetermined signalfrequency.
 13. The particle processing system of claim 12, wherein theradiation source system is configured to sequentially interrogate eachof the plurality of interrogation sites with the predetermined timeddelay, wherein the radiation source system includes a single radiationsource, and wherein the predetermined timed delay is generated byproviding different optical path lengths between the single radiationsource and each of the plurality of interrogation sites.
 14. Theparticle processing system of claim 12, wherein the radiation sourcesystem is configured to sequentially interrogate each of the pluralityof interrogation sites with the predetermined timed delay, wherein theradiation source system includes a single radiation source, and whereinthe predetermined timed delay is generated by providing differentoptical path refraction indexes between the single radiation source andeach of the plurality of interrogation sites.
 15. The particleprocessing system of claim 12, wherein the radiation source system isconfigured to sequentially interrogate each of the plurality ofinterrogation sites with the predetermined timed delay, the particleprocessing system further comprising: a controller configured tosynchronize the predetermined timed delay with the optical signalsreceived by the detector.
 16. The particle processing system of claim12, wherein the radiation source system is configured to sequentiallyinterrogate each of the plurality of interrogation sites with thepredetermined timed delay, wherein the radiation source system includesa single radiation source, and wherein the predetermined timed delay isgenerated by providing different optical path lengths between the singleradiation source and each of the plurality of interrogation sites. 17.(canceled)
 18. The particle processing system of claim 12, wherein theradiation source system is configured to simultaneously interrogate afirst interrogation region with a first predetermined signal frequencyand a second interrogation region with a second predetermined signalfrequency, the particle processing system further comprising anelectronic system configured to separate the optical signals from theplurality of interrogation sites based on a recognition of thepredetermined signal frequencies.
 19. A particle processing systemcomprising: a radiation source system configured to opticallyinterrogate a plurality of interrogation sites; a signal detectionsystem including a detector configured to receive optical signalsemitted from the plurality of interrogation sites; a filter arrayprovided as at least one of a spatial filter array and a spectral filterarray, the filter array providing a distinctive pattern for eachinterrogation site.
 20. The particle processing system of claim 19,wherein the filter array is a spatial filter array.
 21. The particleprocessing system of claim 19, wherein the filter array is a spectralfilter array.
 22. The particle processing system of claim 19, furthercomprising an electronic system configured to separate the opticalsignals from the plurality of interrogation sites based on a recognitionof the distinctive pattern for each interrogation site.